Activin-actriia antagonists for inhibiting germ cell maturation

ABSTRACT

In certain aspects, the present invention provides compositions and methods for decreasing FSH levels in a patient. The patient may, for example, be diagnosed with an FSH-related disorder or desire to delay or inhibit germ cell maturation.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. application Ser. No.13/111,393, filed May 19, 2011, which is a divisional of U.S.application Ser. No. 12/284,112, filed Sep. 17, 2008 (now U.S. Pat. No.7,960,343), which claims the benefit of U.S. Provisional ApplicationSer. No. 60/994,399, filed Sep. 18, 2007. The specifications of each ofthe foregoing applications are incorporated herein by reference in theirentirety.

SEQUENCE LISTING

The instant application contains a Sequence Listing which has beensubmitted via EFS-Web and is hereby incorporated by reference in itsentirety. Said ASCII copy, created on Jan. 30, 2013, is namedPHPH028103_Seq.txt, and is 24,693 bytes in size.

BACKGROUND OF THE INVENTION

Follicle-stimulating hormone (FSH) is released by the pituitary glandand regulates the functioning of the gonads and the production andmaturation of gametes. FSH is generally released by the pituitary glandupon prior release of a triggering hormone, such asgonadotropin-releasing hormone.

FSH release is necessary for ovulation in females and for maturation ofsperm in males. In females, FSH stimulates follicular granulosa cellproliferation in the ovary and impacts synthesis of estrogen, a hormonewhich is integral to follicular maturation and ovulation. In males, FSHis involved in the maturation of sperm cells. More specifically, FSHaction in males is directed at the Sertoli cells, which are a recognizedtarget of the hormone and which support the process of sperm maturation(spermatogenesis). FSH is also produced in the prostate, where it is animportant mediator of cell growth.

Accordingly, inhibitors of FSH release are useful as contraceptiveagents in both males and females.

In addition to the function in fertility, FSH also plays a role inseveral disease states. Increased levels of FSH receptor are associatedwith prostate cancer, with the highest levels associated withhormone-refractory prostate cancer. Prostate cancer is the most commoncancer in American men, with more than 230,000 new cases diagnosed eachyear. Approximately 30,000 deaths will be attributed to prostate cancerin 2004 (Jemal A, Tiwari R C, Murray T. Ghafoor A, Samuels A, Ward E,Feuer E J, Thun M J. Cancer statistics 2004. CA Cancer J. Clin. 54:8-29,2004). Approximately 40% of individuals treated with surgery orradiation will develop recurrent prostate cancer (Walsh P C, Retik A B,Vaughan E D, eds. Campbell's Urology. 7th ed. Philadelphia, Pa.: WBSaunders Company; 1998). The most common treatment for recurrentprostate cancer is the suppression of testicular testosterone productionvia orchiectomy, estrogen treatment, antiandrogen administration, and/orGnRH agonist/antagonist treatment. This usually results in remission for2-3 years, after which time prostate cancer becomes “hormonerefractory,” meaning that it develops the ability to grow despite thereduction of blood androgen concentrations to castrate levels.Consequently, improved compositions and methods are needed for treatingprostate cancer, in particular hormone refractory prostate cancer.

Pituitary tumors (adenoma) are non-cancerous growths that typicallyaffect different hormone-producing regions, depending on the specificlocation of the tumor. Pituitary tumors account for about 15% ofintracranial tumors, and are associated with significant morbibity dueto local compressive effects, hormonal hypersecretion, ortreatment-associated endocrine deficiency (Heaney A. P., et al.:Molecular Pathogenesis of Pituitary Tumors. In: Oxford Textbook ofEndocrinology, Wass J. A. H. and Shalet S. M., (Eds.), Oxford UniversityPress, Oxford, 2002 (in press)). The great majority of pituitaryadenomas are benign and are relatively slow growing. Pituitary tumorsmay, however, lead to overproduction of one or more of the pituitaryhormones. FSH-secreting pituitary tumors often lead to the developmentof multicystic ovaries and to elevated estradiol levels. In turn,increases in estradiol levels contribute to health risks includingendometrial and prostate cancer. Consequently, improved compositions andmethods are needed for treating symptoms associated with FSH-secretingpituitary tumors.

Accordingly, compounds that inhibit FSH secretion are useful in avariety of treatments.

It is an object of the present disclosure to provide compositions andmethods that may be used to decrease FSH levels, and such compositionsand methods may be used, for example, in contraception and for thetreatment of a variety of FSH-related disorders.

SUMMARY OF THE INVENTION

In part, the disclosure relates to the use of activin antagonists, aswell as ActRIIa antagonists, to decrease or inhibit FSH secretion. Inparticular, the disclosure provides methods for decreasing or inhibitingFSH secretion using a soluble form of ActRIIa that acts as an inhibitorof activin. While soluble ActRIIa may affect FSH secretion through amechanism other than activin antagonism, desirable therapeutic agentsmay nonetheless be selected on the basis of activin antagonism orActRIIa antagonism or both. Such agents are referred to collectively asactivin-ActRIIa antagonists. Therefore, in certain embodiments, thedisclosure provides methods for using activin-ActRIIa antagonists,including, for example, activin-binding ActRIIa polypeptides,anti-activin antibodies, anti-ActRIIa antibodies, activin- orActRIIa-targeted small molecules and aptamers, and nucleic acids thatdecrease expression of activin and ActRIIa, to decrease or inhibit FSHsecretion in patients in need thereof. As described in U.S. PublicationNo. 2007/0249022, incorporated by reference herein, activin-ActRIIaantagonists can be used to promote bone growth and increase bonedensity. As described herein, such antagonists can also be used todecrease or inhibit FSH secretion.

In certain aspects, the disclosure provides methods for decreasing orinhibiting FSH secretion using polypeptides comprising a soluble,activin-binding ActRIIa polypeptide that binds to activin. ActRIIapolypeptides may be formulated as a pharmaceutical preparationcomprising the activin-binding ActRIIa polypeptide and apharmaceutically acceptable carrier. The activin-binding ActRIIapolypeptide may bind to activin with a K_(D) less than 1 micromolar orless than 100, 10 or 1 nanomolar. Optionally, the activin-bindingActRIIa polypeptide selectively binds activin versus GDF11 and/or GDF8,and optionally with a K_(D) that is at least 10-fold, 20-fold or 50-foldlower with respect to activin than with respect to GDF11 and/or GDF8.While not wishing to be bound to a particular mechanism of action, it isexpected that this degree of selectivity for activin inhibition overGDF11/GDF8 inhibition accounts for effects on FSH secretion without aconsistently measurable effect on muscle. In many embodiments, anActRIIa polypeptide will be selected for causing less than 15%, lessthan 10% or less than 5% increase in muscle at doses that achievedesirable effects on FSH secretion. The composition may be at least 95%pure, with respect to other polypeptide components, as assessed by sizeexclusion chromatography, and optionally, the composition is at least98% pure. An activin-binding ActRIIa polypeptide for use in such apreparation may be any of those disclosed herein, such as a polypeptidehaving an amino acid sequence selected from SEQ ID NOs: 2, 3, 7 or 12,or having an amino acid sequence that is at least 80%, 85%, 90%, 95%,97% or 99% identical to an amino acid sequence selected from SEQ ID NOs:2, 3, 7, 12 or 13. An activin-binding ActRIIa polypeptide may include afunctional fragment of a natural ActRIIa polypeptide, such as onecomprising at least 10, 20 or 30 amino acids of a sequence selected fromSEQ ID NOs: 1-3 or a sequence of SEQ ID NO: 2, lacking the C-terminal 10to 15 amino acids (the “tail”).

In certain aspects, the disclosure provides methods for decreasing FSHlevels in a human subject having an FSH-related disorder. Such a methodmay comprise administering to the subject an amount of an ActRIIa-Fcfusion protein effective to reduce FSH activity in the subject. Incertain aspects, the disclosure provides methods for decreasing FSHlevels in a patient desiring to delay or inhibit his or her germ cellmaturation. Such a method may comprise administering an amount ofActRIIa-Fc fusion protein effective to reduce FSH activity in thesubject. ActRIIa-Fc fusion protein may comprises an amino acid sequencethat is at least 90%, 95%, 98%, 99% or 100% identical to the amino acidsequence of SEQ ID NO:3 or SEQ ID NO:2. The ActRIIa-Fc fusion proteinmay be a dimer formed of two polypeptides that each comprise an aminoacid sequence that is at least 90%, 95%, 98%, 99% or 100% identical tothe amino acid sequence of SEQ ID NO:3 or SEQ ID NO:2. The ActRIIa-Fcfusion protein may comprise three or more sialic acid moieties,particularly three, four or five sialic acid moieties. The ActRIIa-Fcfusion protein may be produced in CHO cells. The ActRIIa-Fc fusionprotein may have an amino acid sequence of SEQ ID NO:7. The ActRIIa-Fcfusion protein may be administered so as to reach a serum concentrationin the patient of at least 0.3 mg/kg, and preferably to reach a serumconcentration ranging between 0.3 and 3 mg/kg. The ActRIIa-Fc fusionprotein may have a serum half-life of between 15 and 30 days and may,for example, be administered to the subject no more frequently than onceper week, once per month or once per year. In a certain embodiment, theActRIIa-Fc fusion protein has a serum half-life of 25 to 32 days onaverage in normal, healthy humans and equivalent bioavailability whenadministered intravenously or subcutaneously. The ActRIIa-Fc fusionprotein may be administered intravenously or subcutaneously.

A soluble, activin-binding ActRIIa polypeptide may include one or morealterations in the amino acid sequence (e.g., in the ligand-bindingdomain) relative to a naturally occurring ActRIIa polypeptide. Examplesof altered ActRIIa polypeptides are provided in WO 2006/012627, pp.59-60, incorporated by reference herein. The alteration in the aminoacid sequence may, for example, alter glycosylation of the polypeptidewhen produced in a mammalian, insect or other eukaryotic cell or alterproteolytic cleavage of the polypeptide relative to the naturallyoccurring ActRIIa polypeptide.

An activin-binding ActRIIa polypeptide may be a fusion protein that has,as one domain, an ActRIIa polypeptide (e.g., a ligand-binding portion ofan ActRIIa) and one or more additional domains that provide a desirableproperty, such as improved pharmacokinetics, easier purification,targeting to particular tissues, etc. For example, a domain of a fusionprotein may enhance one or more of in vivo stability, in vivo half life,uptake/administration, tissue localization or distribution, formation ofprotein complexes, multimerization of the fusion protein, and/orpurification. An activin-binding ActRIIa fusion protein may include animmunoglobulin Fc domain (wild-type or mutant) or a serum albumin orother polypeptide portion that provides desirable properties such asimproved pharmacokinetics, improved solubility or improved stability. Ina preferred embodiment, an ActRIIa-Fc fusion comprises a relativelyunstructured linker positioned between the Fc domain and theextracellular ActRIIa domain. This unstructured linker may correspond tothe roughly 15 amino acid unstructured region at the C-terminal end ofthe extracellular domain of ActRIIa (the “tail”), or it may be anartificial sequence of 1, 2, 3, 4 or 5 amino acids or a length ofbetween 5 and 15, 20, 30, 50 or more amino acids that are relativelyfree of secondary structure, or a mixture of both. A linker may be richin glycine and proline residues and may, for example, contain a singlesequence of threonine/serine and glycines or repeating sequences ofthreonine/serine and glycines (e.g., TG₄ (SEQ ID NO: 15) or SG₄ (SEQ IDNO: 16) singlets or repeats). A fusion protein may include apurification subsequence, such as an epitope tag, a FLAG tag, apolyhistidine sequence, and a GST fusion. Optionally, a soluble ActRIIapolypeptide includes one or more modified amino acid residues selectedfrom: a glycosylated amino acid, a PEGylated amino acid, a farnesylatedamino acid, an acetylated amino acid, a biotinylated amino acid, anamino acid conjugated to a lipid moiety, and an amino acid conjugated toan organic derivatizing agent. Preferably, a pharmaceutical preparationis substantially pyrogen free. In general, it is preferable that anActRIIa protein be expressed in a mammalian cell line that mediatessuitably natural glycosylation of the ActRIIa protein so as to diminishthe likelihood of an unfavorable immune response in a patient. Human andCHO cell lines have been used successfully, and it is expected thatother common mammalian expression systems will be useful.

As described herein, ActRIIa proteins designated ActRIIa-Fc (a form witha minimal linker between the ActRIIa portion and the Fc portion) havedesirable properties, including selective binding to activin versus GDF8and/or GDF11, high affinity ligand binding and serum half life greaterthan two weeks in animal models. In certain embodiments the inventionprovides methods for decreasing or inhibiting FSH secretion usingActRIIa-Fc polypeptides and pharmaceutical preparations comprising suchpolypeptides and a pharmaceutically acceptable excipient.

In certain aspects, the disclosure provides methods for decreasing orinhibiting FSH secretion using nucleic acids encoding a solubleactivin-binding ActRIIa polypeptide. An isolated polynucleotide maycomprise a coding sequence for a soluble, activin-binding ActRIIapolypeptide, such as described above. For example, an isolated nucleicacid may include a sequence coding for an extracellular domain (e.g.,ligand-binding domain) of an ActRIIa and a sequence that would code forpart or all of the transmembrane domain and/or the cytoplasmic domain ofan ActRIIa, but for a stop codon positioned within the transmembranedomain or the cytoplasmic domain, or positioned between theextracellular domain and the transmembrane domain or cytoplasmic domain.For example, an isolated polynucleotide may comprise a full-lengthActRIIa polynucleotide sequence such as SEQ ID NO: 4 or 5, or apartially truncated version, said isolated polynucleotide furthercomprising a transcription termination codon at least six hundrednucleotides before the 3′-terminus or otherwise positioned such thattranslation of the polynucleotide gives rise to an extracellular domainoptionally fused to a truncated portion of a full-length ActRIIa. Apreferred nucleic acid sequence is SEQ ID NO:14. Nucleic acids useful inaccordance with the methods described herein may be operably linked to apromoter for expression, and the disclosure provides cells transformedwith such recombinant polynucleotides. Preferably the cell is amammalian cell such as a CHO cell.

The disclosure also provides methods for making a soluble,activin-binding ActRIIa polypeptide that can be used for decreasing orinhibiting FSH secretion. Such a method may include expressing any ofthe nucleic acids (e.g., SEQ ID NO: 4, 5 or 14) disclosed herein in asuitable cell, such as a Chinese hamster ovary (CHO) cell. Such a methodmay comprise: a) culturing a cell under conditions suitable forexpression of the soluble ActRIIa polypeptide, wherein said cell istransformed with a soluble ActRIIa expression construct; and b)recovering the soluble ActRIIa polypeptide so expressed. Soluble ActRIIapolypeptides may be recovered as crude, partially purified or highlypurified fractions. Purification may be achieved by a series ofpurification steps, including, for example, one, two or three or more ofthe following, in any order: protein A chromatography, anion exchangechromatography (e.g., Q sepharose), hydrophobic interactionchromatography (e.g., phenylsepharose), size exclusion chromatography,and cation exchange chromatography.

In certain aspects, an activin-ActRIIa antagonist disclosed herein, suchas a soluble, activin-binding ActRIIa polypeptide, may be used in amethod for decreasing or inhibiting FSH secretion in a subject,including, for example, methods for delaying the onset of prostatecancer, inhibiting the progression of prostrate cancer, reducing tumorsize, preventing tumor growth, delaying the onset of metastasis orpreventing metastasis. In certain embodiments, the disclosure providesmethods for decreasing or inhibiting the growth or survival of prostatecancer cells in patients in need thereof. A method may compriseadministering to a subject in need thereof an effective amount ofactivin-ActRIIa antagonist. In certain aspects, the disclosure providesuses of activin-ActRIIa antagonists for making a medicament for thetreatment or prevention of prostate cancer as described herein. Thedisclosure also relates to combination therapies comprising anactivin-ActRIIa antagonist and radiation therapy, chemotherapy (e.g., acytotoxic agent), and/or endocrine therapy. The antagonist may be anActRIIa-Fc fusion protein, wherein the ActRIIa-Fc fusion proteincomprises an amino acid sequence that is at least 90% identical to theamino acid sequence of SEQ ID NO:3, 6, 7, or 13.

In further embodiments, the present invention relates to methods ofpreventing or delaying the onset of prostate cancer in patients with oneor more prostate cancer risk factors. In some embodiments, the inventionrelates to methods of preventing or delaying the onset of metastaticdisease in patients already diagnosed with a primary prostate tumor orwith a proliferative lesion of the prostate. The method of preventing ordelaying the onset of prostate cancer in a human patient may compriseadministering to a human patient in need thereof an effective amount ofa polypeptide selected from the group consisting of: a) a polypeptidecomprising an amino acid sequence at least 90% identical to SEQ ID NO:2;b) a polypeptide comprising an amino acid sequence at least 90%identical to SEQ ID NO:3; and c) a polypeptide comprising at least 50consecutive amino acids selected from SEQ ID NO: 2.

Other embodiments of the invention relate to a method of inhibitingactivin-mediated signaling in a human patient with prostate cancer. Incertain embodiments, the method comprises administering to the humanpatient an effective amount of an activin-ActRIIa antagonist. In furtherembodiments, the antagonist is a polypeptide selected from the groupconsisting of: a) a polypeptide comprising an amino acid sequence atleast 90% identical to SEQ ID NO:2; b) a polypeptide comprising an aminoacid sequence at least 90% identical to SEQ ID NO:3; and c) apolypeptide comprising at least 50 consecutive amino acids selected fromSEQ ID NO: 2.

In certain embodiments, the decrease or inhibition of FSH secretioncauses a reduction in fertility. In females, administration ofactivin-ActRII antagonists limit proliferation of follicular granulosacells. In males, administration of activin-ActRII antagonists inhibitssperm maturation. In certain aspects, the disclosure provides methodsand compositions for contraceptives. In certain embodiments,compositions are provided comprising activin-ActRII antagonists and oneor more oral contraceptive agents, such as progestin, progesterone, andestrogen.

In certain embodiments, methods are provided for decreasing orinhibiting FSH secretions in patients afflicted with FSH-secretingpituitary tumor; the methods comprising administering activin-ActRIIantagonists.

In certain aspects, the disclosure provides a method for identifying anagent that inhibits the growth or survival of cancer cells (e.g.,prostate cancer cells). The method comprises: a) identifying a testagent that binds to activin or a ligand-binding domain of an ActRIIapolypeptide; and b) evaluating the effect of the agent on theproliferation, survival, or apoptosis of cancer cells.

BRIEF DESCRIPTION OF THE DRAWINGS

The patent or application file contains at least one drawing executed incolor. Copies of this patent or patent application publication withcolor drawing(s) will be provided by the Office upon request and paymentof the necessary fee.

FIG. 1 shows the purification of ActRIIa-hFc expressed in CHO cells. Theprotein purifies as a single, well-defined peak.

FIG. 2 shows the binding of ActRIIa-hFc to activin and GDF-11, asmeasured by BiaCore™ assay.

FIG. 3 shows a schematic for the A-204 Reporter Gene Assay. The figureshows the Reporter vector: pGL3(CAGA)12 (described in Dennler et al,1998, EMBO 17: 3091-3100.) The CAGA12 motif is present in TGF-Betaresponsive genes (PAI-1 gene), so this vector is of general use forfactors signaling through Smad 2 and 3.

FIG. 4 shows the effects of ActRIIa-hFc (diamonds) and ActRIIa-mFc(squares) on GDF-8 signaling in the A-204 Reporter Gene Assay. Bothproteins exhibited substantial inhibition of GDF-8 mediated signaling atpicomolar concentrations.

FIG. 5 shows the effects of three different preparations of ActRIIa-hFcon GDF-11 signaling in the A-204 Reporter Gene Assay.

FIG. 6 shows examples of DEXA images of control- and ActRIIa-mFc-treatedBALB/c mice, before (top panels) and after (bottom panels) the 12-weektreatment period. Paler shading indicates increased bone density.

FIG. 7 shows a quantification of the effects of ActRIIa-mFc on bonemineral density in BALB/c mice over the 12-week period. Treatments werecontrol (diamonds), 2 mg/kg dosing of ActRIIa-mFc (squares), 6 mg/kgdosing of ActRIIa-mFc (triangles) and 10 mg/kg dosing of ActRIIa-mFc(circles).

FIG. 8 shows a quantification of the effects of ActRIIa-mFc on bonemineral content in BALB/c mice over the 12-week period. Treatments werecontrol (diamonds), 2 mg/kg dosing of ActRIIa-mFc (squares), 6 mg/kgdosing of ActRIIa-mFc (triangles) and 10 mg/kg dosing of ActRIIa-mFc(circles).

FIG. 9 shows a quantification of the effects of ActRIIa-mFc on bonemineral density of the trabecular bone in ovariectomized (OVX) or shamoperated (SHAM) C57BL6 mice over after a 6-week period. Treatments werecontrol (PBS) or 10 mg/kg dosing of ActRIIa-mFc (ActRIIa).

FIG. 10 shows a quantification of the effects of ActRIIa-mFc on thetrabecular bone in ovariectomized (OVX) C57BL6 mice over a 12-weekperiod. Treatments were control (PBS; pale bars) or 10 mg/kg dosing ofActRIIa-mFc (ActRIIa; dark bars).

FIG. 11 shows a quantification of the effects of ActRIIa-mFc on thetrabecular bone in sham operated C57BL6 mice after 6 or 12 weeks oftreatment period. Treatments were control (PBS; pale bars) or 10 mg/kgdosing of ActRIIa-mFc (ActRIIa; dark bars).

FIG. 12 shows the results of pQCT analysis of bone density inovariectomized mice over 12 weeks of treatment. Treatments were control(PBS; pale bars) or ActRIIa-mFc (dark bars). y-axis: mg/ccm

FIG. 13 depicts the results of pQCT analysis of bone density in shamoperated mice over 12 weeks of treatment. Treatments were control (PBS;pale bars) or ActRIIa-mFc (dark bars). y-axis; mg/ccm

FIGS. 14A and 14B show whole body DEXA analysis after 12 weeks oftreatment (A) and ex vivo analysis of femurs (B). Light areas depictareas of high bone density.

FIG. 15 shows ex vivo pQCT analysis of the femoral midshaft after twelveweeks of treatment. Treatments were vehicle control (PBS, dark bars) andActRIIa-mFc (pale bars). The four bars to the left show total bonedensity while the four bars to the right show cortical bone density. Thefirst pair of bars in each set of four bars represent data fromovariectomized mice while the second pair of bars represent data fromsham operated mice.

FIG. 16 shows ex vivo pQCT analysis and diaphyseal bone content of thefemoral midshaft after twelve weeks of treatment. Treatments werevehicle control (PBS, dark bars) or ActRIIa-mFc (pale bars). The fourbars to the left show total bone content while the four bars to theright show cortical bone content. The first pair of bars in each set offour bars represent data from ovariectomized mice while the second pairof bars represent data from sham operated mice.

FIG. 17 shows ex vivo pQCT analysis of the femoral midshaft and femoralcortical thickness. Treatments were control (PBS, dark bars) andActRIIa-mFc (pale bars). The four bars to the left show endostealcircumference while the four bars to the right show periostealcircumference. The first pair of bars in each set of four bars representdata from ovariectomized mice while the second pair of bars representdata from sham operated mice.

FIG. 18 depicts the results of mechanical testing of femurs after twelveweeks of treatment. Treatments were control (PBS, dark bars) andActRIIa-mFc (pale bars). The two bars to the left represent data fromovariectomized mice while the last two bars represent data from shamoperated mice.

FIG. 19 shows the effects of ActrIIa-mFc on trabecular bone volume.

FIG. 20 shows the effects of ActrIIa-mFc on trabecular architecture inthe distal femur.

FIG. 21 shows the effects of ActrIIa-mFc on cortical bone.

FIG. 22 shows the effects of ActrIIa-mFc on the mechanical strength ofbone.

FIG. 23 shows the effects of different doses of ActRIIa-mFc on bonecharacteristics at three different dosages.

FIG. 24 shows bone histomorphometry indicating that ActRIIa-mFc has dualanabolic and anti-resorptive activity.

FIG. 25 shows additional histomorphometric data.

FIG. 26 shows images of mouse femurs from naïve and tumor-carrying mice,and the effects of ActRIIa-mFc treatment on bone morphology in themultiple myeloma model. Mice carrying multiple myeloma tumors (5T2) showmarked pitting and degradation in the bone relative to normal mice(naïve). Treatment with ActRIIa-mFc eliminates this effect.

FIG. 27 shows results from the human clinical trial described in Example6, where the area-under-curve (AUC) and administered dose of ActRIIa-hFchave a linear correlation, regardless of whether ActRIIa-hFc wasadministered intravenously (IV) or subcutaneously (SC).

FIG. 28 shows a comparison of serum levels of ActRIIa-hFc in patientsadministered IV or SC.

FIG. 29 shows bone alkaline phosphatase (BAP) levels in response todifferent dose levels of ActRIIa-hFc. BAP is a marker for anabolic bonegrowth.

FIG. 30 shows the cooperative effects of ActRIIa-mFc (RAP-011) and abisphosphonate agent (zoledronate) in mice.

FIG. 31 shows results from the human clinical trial described in Example6, showing that ActRIIa-hFc decreases FSH levels in a time- anddose-dependent manner.

FIG. 32 shows an AUC analysis for the dose of ActRIIa-hFc that achievesvarying degrees of effect on FSH levels.

DETAILED DESCRIPTION OF THE INVENTION 1. Overview

The transforming growth factor-beta (TGF-beta) superfamily contains avariety of growth factors that share common sequence elements andstructural motifs. These proteins are known to exert biological effectson a large variety of cell types in both vertebrates and invertebrates.Members of the superfamily perform important functions during embryonicdevelopment in pattern formation and tissue specification and caninfluence a variety of differentiation processes, includingadipogenesis, myogenesis, chondrogenesis, cardiogenesis, hematopoiesis,neurogenesis, and epithelial cell differentiation. The family is dividedinto two general branches: the BMP/GDF and the TGF-beta/Activinbranches, whose members have diverse, often complementary effects. Bymanipulating the activity of a member of the TGF-beta family, it isoften possible to cause significant physiological changes in anorganism. For example, the Piedmontese and Belgian Blue cattle breedscarry a loss-of-function mutation in the GDF8 (also called myostatin)gene that causes a marked increase in muscle mass. Grobet et al., NatGenet. 1997, 17(1):71-4. Furthermore, in humans, inactive alleles ofGDF8 are associated with increased muscle mass and, reportedly,exceptional strength. Schuelke et al., N Engl J Med 2004, 350:2682-8.

Activins are dimeric polypeptide growth factors that belong to theTGF-beta superfamily. There are three principal activin forms (A, B, andAB) that are homo/heterodimers of two closely related β subunits(β_(A)β_(A), β_(B)β_(B), and β_(A)β_(B), respectively). The human genomealso encodes an activin C and an activin E, which are primarilyexpressed in the liver, and heterodimeric forms containing β_(C) orβ_(E) are also known. In the TGF-beta superfamily, activins are uniqueand multifunctional factors that can stimulate hormone production inovarian and placental cells, support neuronal cell survival, influencecell-cycle progress positively or negatively depending on cell type, andinduce mesodermal differentiation at least in amphibian embryos (DePaoloet al., 1991, Proc Soc Ep Biol Med. 198:500-512; Dyson et al., 1997,Curr Biol. 7:81-84; Woodruff, 1998, Biochem Pharmacol. 55:953-963). Inseveral tissues, activin signaling is antagonized by its relatedheterodimer, inhibin. For example, during the release offollicle-stimulating hormone (FSH) from the pituitary, activin promotesFSH secretion and synthesis, while inhibin prevents FSH secretion andsynthesis. Other proteins that may regulate activin bioactivity and/orbind to activin include follistatin (FS), follistatin-related protein(FSRP) and α₂-macroglobulin.

TGF-β signals are mediated by heteromeric complexes of type I and typeII serine/threonine kinase receptors, which phosphorylate and activatedownstream Smad proteins upon ligand stimulation (Massagué, 2000, Nat.Rev. Mol. Cell Biol. 1:169-178). These type I and type II receptors aretransmembrane proteins, composed of a ligand-binding extracellulardomain with cysteine-rich region, a transmembrane domain, and acytoplasmic domain with predicted serine/threonine specificity. Type Ireceptors are essential for signaling; and type II receptors arerequired for binding ligands and for expression of type I receptors.Type I and II activin receptors form a stable complex after ligandbinding, resulting in phosphorylation of type I receptors by type IIreceptors.

Two related type II receptors, ActRIIa and ActRIIb, have been identifiedas the type II receptors for activins (Mathews and Vale, 1991, Cell65:973-982; Attisano et al., 1992, Cell 68: 97-108). Besides activins,ActRIIa and ActRIIb can biochemically interact with several other TGF-βfamily proteins, including BMP7, Nodal, GDF8, and GDF11 (Yamashita etal., 1995, J. Cell Biol. 130:217-226; Lee and McPherron, 2001, Proc.Natl. Acad. Sci. 98:9306-9311; Yeo and Whitman, 2001, Mol. Cell 7:949-957; Oh et al., 2002, Genes Dev. 16:2749-54). ALK4 is the primarytype I receptor for activins, particularly for activin A, and ALK-7 mayserve as a receptor for activins as well, particularly for activin B.

As described herein, a soluble ActRIIa polypeptide (sActRIIa), whichshows substantial preference in binding to activin A as opposed to otherTGF-beta family members, such as GDF8 or GDF11, may be used to decreaseor inhibit FSH secretion. While not wishing to be bound to anyparticular mechanism, it is expected that the effect of sActRIIa iscaused primarily by an activin antagonist effect, given the very strongactivin binding (picomolar dissociation constant) exhibited by theparticular sActRIIa construct used in these studies. Activin-ActRIIaantagonists include, for example, activin-binding soluble ActRIIapolypeptides, antibodies that bind to activin (particularly the activinA or B subunits, also referred to as βA or βB) and disrupt ActRIIabinding, antibodies that bind to ActRIIa and disrupt activin binding,non-antibody proteins selected for activin or ActRIIa binding (see e.g.,WO/2002/088171, WO/2006/055689, and WO/2002/032925 for examples of suchproteins and methods for design and selection of same), randomizedpeptides selected for activin or ActRIIa binding, often affixed to an Fcdomain. Two different proteins (or other moieties) with activin orActRIIa binding activity, especially activin binders that block the typeI (e.g., a soluble type I activin receptor) and type II (e.g., a solubletype II activin receptor) binding sites, respectively, may be linkedtogether to create a bifunctional binding molecule. Nucleic acidaptamers, small molecules and other agents that inhibit theactivin-ActRIIa signaling axis. Various proteins have activin-ActRIIaantagonist activity, including inhibin (i.e., inhibin alpha subunit),although inhibin does not universally antagonize activin in all tissues,follistatin (e.g., follistatin-288 and follistatin-315), FSRP, activinC, alpha(2)-macroglobulin, and an M108A (methionine to alanine change atposition 108) mutant activin A. Generally, alternative forms of activin,particularly those with alterations in the type I receptor bindingdomain can bind to type II receptors and fail to form an active ternarycomplex, thus acting as antagonists. Additionally, nucleic acids, suchas antisense molecules, siRNAs or ribozymes that inhibit activin A, B, Cor E, or, particularly, ActRIIa expression, can be used asactivin-ActRIIa antagonists. The activin-ActRIIa antagonist to be usedmay exhibit selectivity for inhibiting activin-mediated signaling versusother members of the TGF-beta family, and particularly with respect toGDF8 and GDF11. Soluble ActRIIb proteins do bind to activin, however,the wild type protein does not exhibit significant selectivity inbinding to activin versus GDF8/11. Nonetheless, such ActRIIbpolypeptides, as well as altered forms of ActRIIb with different bindingproperties (see, e.g., WO 2006/012627, pp. 55-59, incorporated herein byreference) may achieve the desired effects on cancer cells. Native oraltered ActRIIb may be given added specificity for activin by couplingwith a second, activin-selective binding agent.

The terms used in this specification generally have their ordinarymeanings in the art, within the context of this invention and in thespecific context where each term is used. Certain terms are discussedbelow or elsewhere in the specification, to provide additional guidanceto the practitioner in describing the compositions and methods of theinvention and how to make and use them. The scope or meaning of any useof a term will be apparent from the specific context in which the termis used.

“About” and “approximately” shall generally mean an acceptable degree oferror for the quantity measured given the nature or precision of themeasurements. Typically, exemplary degrees of error are within 20percent (%), preferably within 10%, and more preferably within 5% of agiven value or range of values.

Alternatively, and particularly in biological systems, the terms “about”and “approximately” may mean values that are within an order ofmagnitude, preferably within 5-fold and more preferably within 2-fold ofa given value. Numerical quantities given herein are approximate unlessstated otherwise, meaning that the term “about” or “approximately” canbe inferred when not expressly stated.

The methods of the invention may include steps of comparing sequences toeach other, including wild-type sequence to one or more mutants(sequence variants). Such comparisons typically comprise alignments ofpolymer sequences, e.g., using sequence alignment programs and/oralgorithms that are well known in the art (for example, BLAST, FASTA andMEGALIGN, to name a few). The skilled artisan can readily appreciatethat, in such alignments, where a mutation contains a residue insertionor deletion, the sequence alignment will introduce a “gap” (typicallyrepresented by a dash, or “A”) in the polymer sequence not containingthe inserted or deleted residue.

“Homologous,” in all its grammatical forms and spelling variations,refers to the relationship between two proteins that possess a “commonevolutionary origin,” including proteins from superfamilies in the samespecies of organism, as well as homologous proteins from differentspecies of organism. Such proteins (and their encoding nucleic acids)have sequence homology, as reflected by their sequence similarity,whether in terms of percent identity or by the presence of specificresidues or motifs and conserved positions.

The term “sequence similarity,” in all its grammatical forms, refers tothe degree of identity or correspondence between nucleic acid or aminoacid sequences that may or may not share a common evolutionary origin.

However, in common usage and in the instant application, the term“homologous,” when modified with an adverb such as “highly,” may referto sequence similarity and may or may not relate to a commonevolutionary origin.

The term “prostate cancer” refers to any proliferative lesion orproliferative abnormality of the prostate including, for example, benignlesions, pre-malignant and malignant lesions, solid tumors, andmetastatic disease (both locally metastatic, e.g., stage III, and morewidely metastatic, e.g., stage IV). Prostate cancer also encompassesboth hormone-responsive and hormone-independent cancers.Hormone-refractory prostate cancers are refractory to treatment withantihormonal (especially antiestrogenic) therapies.

2. ActRIIa Polypeptides

In certain aspects, the present invention relates to ActRIIapolypeptides. As used herein, the term “ActRIIa” refers to a family ofactivin receptor type Ha (ActRIIa) proteins from any species andvariants derived from such ActRIIa proteins by mutagenesis or othermodification. Reference to ActRIIa herein is understood to be areference to any one of the currently identified forms. Members of theActRIIa family are generally transmembrane proteins, composed of aligand-binding extracellular domain with a cysteine-rich region, atransmembrane domain, and a cytoplasmic domain with predictedserine/threonine kinase activity.

The term “ActRIIa polypeptide” includes polypeptides comprising anynaturally occurring polypeptide of an ActRIIa family member as well asany variants thereof (including mutants, fragments, fusions, andpeptidomimetic forms) that retain a useful activity. For example,ActRIIa polypeptides include polypeptides derived from the sequence ofany known ActRIIa having a sequence at least about 80% identical to thesequence of an ActRIIa polypeptide, and preferably at least 85%, 90%,95%, 97%, 99% or greater identity. For example, an ActRIIa polypeptideof the invention may bind to and inhibit the function of an ActRIIaprotein and/or activin. Preferably, an ActRIIa polypeptide decreases FSHlevels in vivo or in an in vitro assay conducted using pituitary cells.Examples of ActRIIa polypeptides include human ActRIIa precursorpolypeptide (SEQ ID NO: 1) and soluble human ActRIIa polypeptides (e.g.,SEQ ID NOs: 2, 3, 7 and 12).

The human ActRIIa precursor protein sequence is as follows:

(SEQ ID NO: 1) MGAAAKLAFAVFLISCSSGA ILGRSETQECLFFNANWEKDRT N QTGVEPCYGDKDKRRHCFATWK N ISGSIEIVKQGCWLDDINCYDRTDCVEKKDSPEVYFCCCEGNMCNEKFSYFPEMEVTQPTSNPVTPKPPYYNILLYSLVPLMLIAGIVICAFWVYRHHKMAYPPVLVPTQDPGPPPPSPLLGLKPLQLLEVKARGRFGCVWKAQLLNEYVAVKIFPIQDKQSWQNEYEVYSLPGMKHENILQFIGAEKRGTSVDVDLWLITAFHEKGSLSDFLKANVVSWNELCHIAETMARGLAYLHEDIPGLKDGHKPAISHRDIKSKNVLLKNNLTACIADFGLALKFEAGKSAGDTHGQVGTRRYMAPEVLEGAINFQRDAFLRIDMYAMGLVLWELASRCTAADGPVDEYMLPFEEEIGQHPSLEDMQEVVVHKKKRPVLRDYWQKHAGMAMLCETIEECWDHDAEARLSAGCVGERITQMQRLTNIITTEDIVTVVTM VTNVDFPPKESSL

The signal peptide is single underlined; the extracellular domain is inbold and the potential N-linked glycosylation sites are doubleunderlined.

The human ActRIIa soluble (extracellular), processed polypeptidesequence is as follows:

(SEQ ID NO: 2) ILGRSETQECLFFNANWEKDRTNQTGVEPCYGDKDKRRHCFATWKNISGSIEIVKQGCWLDDINCYDRTDCVEKKDSPEVYFCCCEGNMCNEKFSYFPEM EVTQPTSNPVTPKPP 

The C-terminal “tail” of the extracellular domain is underlined. Thesequence with the “tail” deleted (a Δ15 sequence) is as follows:

(SEQ ID NO: 3) ILGRSETQECLFFNANWEKDRTNQTGVEPCYGDKDKRRHCFATWKNISGSIEIVKQGCWLDDINCYDRTDCVEKKDSPEVYFCCCEGNMCNEKFSYFP EM

The nucleic acid sequence encoding human ActRIIa precursor protein is asfollows (nucleotides 164-1705 of Genbank entry NM_001616):

(SEQ ID NO: 4) ATGGGAGCTGCTGCAAAGTTGGCGTTTGCCGTCTTTCTTATCTCCTGTTCTTCAGGTGCTATACTTGGTAGATCAGAAACTCAGGAGTGTCTTTTCTTTAATGCTAATTGGGAAAAAGACAGAACCAATCAAACTGGTGTTGAACCGTGTTATGGTGACAAAGATAAACGGCGGCATTGTTTTGCTACCTGGAAGAATATTTCTGGTTCCATTGAAATAGTGAAACAAGGTTGTTGGCTGGATGATATCAACTGCTATGACAGGACTGATTGTGTAGAAAAAAAAGACAGCCCTGAAGTATATTTTTGTTGCTGTGAGGGCAATATGTGTAATGAAAAGTTTTCTTATTTTCCAGAGATGGAAGTCACACAGCCCACTTCAAATCCAGTTACACCTAAGCCACCCTATTACAACATCCTGCTCTATTCCTTGGTGCCACTTATGTTAATTGCGGGGATTGTCATTTGTGCATTTTGGGTGTACAGGCATCACAAGATGGCCTACCCTCCTGTACTTGTTCCAACTCAAGACCCAGGACCACCCCCACCTTCTCCATTACTAGGGTTGAAACCACTGCAGTTATTAGAAGTGAAAGCAAGGGGAAGATTTGGTTGTGTCTGGAAAGCCCAGTTGCTTAACGAATATGTGGCTGTCAAAATATTTCCAATACAGGACAAACAGTCATGGCAAAATGAATACGAAGTCTACAGTTTGCCTGGAATGAAGCATGAGAACATATTACAGTTCATTGGTGCAGAAAAACGAGGCACCAGTGTTGATGTGGATCTTTGGCTGATCACAGCATTTCATGAAAAGGGTTCACTATCAGACTTTCTTAAGGCTAATGTGGTCTCTTGGAATGAACTGTGTCATATTGCAGAAACCATGGCTAGAGGATTGGCATATTTACATGAGGATATACCTGGCCTAAAAGATGGCCACAAACCTGCCATATCTCACAGGGACATCAAAAGTAAAAATGTGCTGTTGAAAAACAACCTGACAGCTTGCATTGCTGACTTTGGGTTGGCCTTAAAATTTGAGGCTGGCAAGTCTGCAGGCGATACCCATGGACAGGTTGGTACCCGGAGGTACATGGCTCCAGAGGTATTAGAGGGTGCTATAAACTTCCAAAGGGATGCATTTTTGAGGATAGATATGTATGCCATGGGATTAGTCCTATGGGAACTGGCTTCTCGCTGTACTGCTGCAGATGGACCTGTAGATGAATACATGTTGCCATTTGAGGAGGAAATTGGCCAGCATCCATCTCTTGAAGACATGCAGGAAGTTGTTGTGCATAAAAAAAAGAGGCCTGTTTTAAGAGATTATTGGCAGAAACATGCTGGAATGGCAATGCTCTGTGAAACCATTGAAGAATGTTGGGATCACGACGCAGAAGCCAGGTTATCAGCTGGATGTGTAGGTGAAAGAATTACCCAGATGCAGAGACTAACAAATATTATTACCACAGAGGACATTGTAACAGTGGTCACAATGGTGACAAATGTTGACTTTCCTCCCAAAGAATCTAGTCTATGA 

The nucleic acid sequence encoding a human ActRIIa soluble(extracellular) polypeptide is as follows:

(SEQ ID NO: 5) ATACTTGGTAGATCAGAAACTCAGGAGTGTCTTTTCTTTAATGCTAATTGGGAAAAAGACAGAACCAATCAAACTGGTGTTGAACCGTGTTATGGTGACAAAGATAAACGGCGGCATTGTTTTGCTACCTGGAAGAATATTTCTGGTTCCATTGAAATAGTGAAACAAGGTTGTTGGCTGGATGATATCAACTGCTATGACAGGACTGATTGTGTAGAAAAAAAAGACAGCCCTGAAGTATATTTTTGTTGCTGTGAGGGCAATATGTGTAATGAAAAGTTTTCTTATTTTCCAGAGATGGAAGTCACACAGCCCACTTCAAATCCAGTTACACCTAAGCCACCC 

In a specific embodiment, the invention relates to soluble ActRIIapolypeptides and their uses in decreasing FSH levels. As describedherein, the term “soluble ActRIIa polypeptide” generally refers topolypeptides comprising an extracellular domain of an ActRIIa protein.The term “soluble ActRIIa polypeptide,” as used herein, includes anynaturally occurring extracellular domain of an ActRIIa protein as wellas any variants thereof (including mutants, fragments and peptidomimeticforms). An activin-binding ActRIIa polypeptide is one that retains theability to bind to activin, particularly activin AA, AB or BB.Preferably, an activin-binding ActRIIa polypeptide will bind to activinAA with a dissociation constant of 1 nM or less. Amino acid sequences ofhuman ActRIIa precursor protein is provided below. The extracellulardomain of an ActRIIa protein binds to activin and is generally soluble,and thus can be termed a soluble, activin-binding ActRIIa polypeptide.Examples of soluble, activin-binding ActRIIa polypeptides include thesoluble polypeptide illustrated in SEQ ID NOs: 2, 3, 7, 12 and 13. SEQID NO:7 is referred to as ActRIIa-hFc, and is described further in theExamples. Other examples of soluble, activin-binding ActRIIapolypeptides comprise a signal sequence in addition to the extracellulardomain of an ActRIIa protein, for example, the honey bee mellitin leadersequence (SEQ ID NO: 8), the tissue plaminogen activator (TPA) leader(SEQ ID NO: 9) or the native ActRIIa leader (SEQ ID NO: 10). TheActRIIa-hFc polypeptide illustrated in SEQ ID NO:13 uses a TPA leader.

Functionally active fragments of ActRIIa polypeptides can be obtained byscreening polypeptides recombinantly produced from the correspondingfragment of the nucleic acid encoding an ActRIIa polypeptide. Inaddition, fragments can be chemically synthesized using techniques knownin the art such as conventional Merrifield solid phase f-Moc or t-Bocchemistry. The fragments can be produced (recombinantly or by chemicalsynthesis) and tested to identify those peptidyl fragments that canfunction as antagonists (inhibitors) of ActRIIa protein or signalingmediated by activin.

Functionally active variants of ActRIIa polypeptides can be obtained byscreening libraries of modified polypeptides recombinantly produced fromthe corresponding mutagenized nucleic acids encoding an ActRIIapolypeptide. The variants can be produced and tested to identify thosethat can function as antagonists (inhibitors) of ActRIIa protein orsignaling mediated by activin. In certain embodiments, a functionalvariant of the ActRIIa polypeptides comprises an amino acid sequencethat is at least 75% identical to an amino acid sequence selected fromSEQ ID NOs: 2 or 3. In certain cases, the functional variant has anamino acid sequence at least 80%, 85%, 90%, 95%, 97%, 98%, 99% or 100%identical to an amino acid sequence selected from SEQ ID NOs: 2 or 3.

Functional variants may be generated by modifying the structure of anActRIIa polypeptide for such purposes as enhancing therapeutic efficacy,or stability (e.g., ex vivo shelf life and resistance to proteolyticdegradation in vivo). Such modified ActRIIa polypeptides when selectedto retain activin binding, are considered functional equivalents of thenaturally-occurring ActRIIa polypeptides. Modified ActRIIa polypeptidescan also be produced, for instance, by amino acid substitution,deletion, or addition. For instance, it is reasonable to expect that anisolated replacement of a leucine with an isoleucine or valine, anaspartate with a glutamate, a threonine with a serine, or a similarreplacement of an amino acid with a structurally related amino acid(e.g., conservative mutations) will not have a major effect on thebiological activity of the resulting molecule. Conservative replacementsare those that take place within a family of amino acids that arerelated in their side chains. Whether a change in the amino acidsequence of an ActRIIa polypeptide results in a functional homolog canbe readily determined by assessing the ability of the variant ActRIIapolypeptide to produce a response in cells in a fashion similar to thewild-type ActRIIa polypeptide.

In certain embodiments, the present invention contemplates specificmutations of the ActRIIa polypeptides so as to alter the glycosylationof the polypeptide. Such mutations may be selected so as to introduce oreliminate one or more glycosylation sites, such as O-linked or N-linkedglycosylation sites. Asparagine-linked glycosylation recognition sitesgenerally comprise a tripeptide sequence, asparagine-X-threonine (orasparagines-X-serine) (where “X” is any amino acid) which isspecifically recognized by appropriate cellular glycosylation enzymes.The alteration may also be made by the addition of, or substitution by,one or more serine or threonine residues to the sequence of thewild-type ActRIIa polypeptide (for O-linked glycosylation sites). Avariety of amino acid substitutions or deletions at one or both of thefirst or third amino acid positions of a glycosylation recognition site(and/or amino acid deletion at the second position) results innon-glycosylation at the modified tripeptide sequence. Another means ofincreasing the number of carbohydrate moieties on an ActRIIa polypeptideis by chemical or enzymatic coupling of glycosides to the ActRIIapolypeptide. Depending on the coupling mode used, the sugar(s) may beattached to (a) arginine and histidine; (b) free carboxyl groups; (c)free sulfhydryl groups such as those of cysteine; (d) free hydroxylgroups such as those of serine, threonine, or hydroxyproline; (e)aromatic residues such as those of phenylalanine, tyrosine, ortryptophan; or (f) the amide group of glutamine. These methods aredescribed in WO 87/05330 published Sep. 11, 1987, and in Aplin andWriston (1981) CRC Crit. Rev. Biochem., pp. 259-306, incorporated byreference herein. Removal of one or more carbohydrate moieties presenton an ActRIIa polypeptide may be accomplished chemically and/orenzymatically. Chemical deglycosylation may involve, for example,exposure of the ActRIIa polypeptide to the compoundtrifluoromethanesulfonic acid, or an equivalent compound. This treatmentresults in the cleavage of most or all sugars except the linking sugar(N-acetylglucosamine or N-acetylgalactosamine), while leaving the aminoacid sequence intact. Chemical deglycosylation is further described byHakimuddin et al. (1987) Arch. Biochem. Biophys. 259:52 and by Edge etal. (1981) Anal. Biochem. 118:131. Enzymatic cleavage of carbohydratemoieties on ActRIIa polypeptides can be achieved by the use of a varietyof endo- and exo-glycosidases as described by Thotakura et al. (1987)Meth. Enzymol. 138:350. The sequence of an ActRIIa polypeptide may beadjusted, as appropriate, depending on the type of expression systemused, as mammalian, yeast, insect and plant cells may all introducediffering glycosylation patterns that can be affected by the amino acidsequence of the peptide. In general, ActRIIa proteins for use in humanswill be expressed in a mammalian cell line that provides properglycosylation, such as HEK293 or CHO cell lines, although othermammalian expression cell lines, yeast cell lines with engineeredglycosylation enzymes and insect cells are expected to be useful aswell.

This disclosure further contemplates a method of generating mutants,particularly sets of combinatorial mutants of an ActRIIa polypeptide, aswell as truncation mutants; pools of combinatorial mutants areespecially useful for identifying functional variant sequences. Thepurpose of screening such combinatorial libraries may be to generate,for example, ActRIIa polypeptide variants which can act as eitheragonists or antagonist, or alternatively, which possess novel activitiesall together. A variety of screening assays are provided below, and suchassays may be used to evaluate variants. For example, an ActRIIapolypeptide variant may be screened for ability to bind to an ActRIIaligand, to prevent binding of an ActRIIa ligand to an ActRIIapolypeptide or to interfere with signaling caused by an ActRIIa ligand.

The activity of an ActRIIa polypeptide or its variants may also betested in a cell-based or in vivo assay. For example, the effect of anActRIIa polypeptide variant on the expression of genes involved in FSHproduction. This may, as needed, be performed in the presence of one ormore recombinant ActRIIa ligand proteins (e.g., activin), and cells maybe transfected so as to produce an ActRIIa polypeptide and/or variantsthereof, and optionally, an ActRIIa ligand. Likewise, an ActRIIapolypeptide may be administered to a mouse or other animal, and FSHlevels may be assessed. Pituitary cell lines that produce FSH are wellknown and ActRIIa proteins may be tested for efficacy in reducing FSHproduction, particularly in the presence of exogenously suppliedactivin. As another example, the effect of an ActRIIa polypeptidevariant on the proliferation or survival of cancer cells may beassessed. Cancer cells may refer to cells in a living subject that makeup a solid tumor or to cells that have originated from a tumor and thathave spread to other sites within a living subject (i.e., metastaticcells). Additionally, cancer cells may refer to cells obtained orderived from a tumor or cancerous growth and that are cultured in vitro.Cancer cells also encompass cell lines that may be cultivated in vitroor used in animal xenograft studies, for example. Cancer cells alsorefer to cells derived from metastatic cells through cell divisionfollowing metastasis. The cells may be hormone-responsive orhormone-independent. Cancer cell proliferation or survival may beassessed in the presence of one or more recombinant ActRIIa ligandproteins (e.g., activin), and cells may be transfected so as to producean ActRIIa polypeptide and/or variants thereof, and optionally, anActRIIa ligand. Likewise, an ActRIIa polypeptide may be administered toa mouse or other animal, and one or more measurements, such as tumorsize, or the rate of cell proliferation or apoptosis relative to acontrol, may be assessed.

Combinatorially-derived variants can be generated which have a selectiveor generally increased potency relative to a naturally occurring ActRIIapolypeptide. Likewise, mutagenesis can give rise to variants which haveintracellular half-lives dramatically different than the corresponding awild-type ActRIIa polypeptide. For example, the altered protein can berendered either more stable or less stable to proteolytic degradation orother cellular processes which result in destruction of, or otherwiseinactivation of a native ActRIIa polypeptide. Such variants, and thegenes which encode them, can be utilized to alter ActRIIa polypeptidelevels by modulating the half-life of the ActRIIa polypeptides. Forinstance, a short half-life can give rise to more transient biologicaleffects and can allow tighter control of recombinant ActRIIa polypeptidelevels within the patient. In an Fc fusion protein, mutations may bemade in the linker (if any) and/or the Fc portion to alter the half-lifeof the protein.

A combinatorial library may be produced by way of a degenerate libraryof genes encoding a library of polypeptides which each include at leasta portion of potential ActRIIa polypeptide sequences. For instance, amixture of synthetic oligonucleotides can be enzymatically ligated intogene sequences such that the degenerate set of potential ActRIIapolypeptide nucleotide sequences are expressible as individualpolypeptides, or alternatively, as a set of larger fusion proteins(e.g., for phage display).

There are many ways by which the library of potential homologs can begenerated from a degenerate oligonucleotide sequence. Chemical synthesisof a degenerate gene sequence can be carried out in an automatic DNAsynthesizer, and the synthetic genes then be ligated into an appropriatevector for expression. The synthesis of degenerate oligonucleotides iswell known in the art (see for example, Narang, SA (1983) Tetrahedron39:3; Itakura et al., (1981) Recombinant DNA, Proc. 3rd ClevelandSympos. Macromolecules, ed. AG Walton, Amsterdam: Elsevier pp 273-289;Itakura et al., (1984) Annu. Rev. Biochem. 53:323; Itakura et al.,(1984) Science 198:1056; Ike et al., (1983) Nucleic Acid Res. 11:477).Such techniques have been employed in the directed evolution of otherproteins (see, for example, Scott et al., (1990) Science 249:386-390;Roberts et al., (1992) PNAS USA 89:2429-2433; Devlin et al., (1990)Science 249: 404-406; Cwirla et al., (1990) PNAS USA 87: 6378-6382; aswell as U.S. Pat. Nos. 5,223,409, 5,198,346, and 5,096,815).

Alternatively, other forms of mutagenesis can be utilized to generate acombinatorial library. For example, ActRIIa polypeptide variants can begenerated and isolated from a library by screening using, for example,alanine scanning mutagenesis and the like (Ruf et al., (1994)Biochemistry 33:1565-1572; Wang et al., (1994) J. Biol. Chem.269:3095-3099; Balint et al., (1993) Gene 137:109-118; Grodberg et al.,(1993) Eur. J. Biochem. 218:597-601; Nagashima et al., (1993) J. Biol.Chem. 268:2888-2892; Lowman et al., (1991) Biochemistry 30:10832-10838;and Cunningham et al., (1989) Science 244:1081-1085), by linker scanningmutagenesis (Gustin et al., (1993) Virology 193:653-660; Brown et al.,(1992) Mol. Cell Biol. 12:2644-2652; McKnight et al., (1982) Science232:316); by saturation mutagenesis (Meyers et al., (1986) Science232:613); by PCR mutagenesis (Leung et al., (1989) Method Cell Mol Biol1:11-19); or by random mutagenesis, including chemical mutagenesis, etc.(Miller et al., (1992) A Short Course in Bacterial Genetics, CSHL Press,Cold Spring Harbor, N.Y.; and Greener et al., (1994) Strategies in MolBiol 7:32-34). Linker scanning mutagenesis, particularly in acombinatorial setting, is an attractive method for identifying truncated(bioactive) forms of ActRIIa polypeptides.

A wide range of techniques are known in the art for screening geneproducts of combinatorial libraries made by point mutations andtruncations, and, for that matter, for screening cDNA libraries for geneproducts having a certain property. Such techniques will be generallyadaptable for rapid screening of the gene libraries generated by thecombinatorial mutagenesis of ActRIIa polypeptides. The most widely usedtechniques for screening large gene libraries typically comprisescloning the gene library into replicable expression vectors,transforming appropriate cells with the resulting library of vectors,and expressing the combinatorial genes under conditions in whichdetection of a desired activity facilitates relatively easy isolation ofthe vector encoding the gene whose product was detected. Preferredassays include activin binding assays and activin-mediated cellsignaling assays.

In certain embodiments, the ActRIIa polypeptides of the invention mayfurther comprise post-translational modifications in addition to anythat are naturally present in the ActRIIa polypeptides. Suchmodifications include, but are not limited to, acetylation,carboxylation, glycosylation, phosphorylation, lipidation, andacylation. As a result, the modified ActRIIa polypeptides may containnon-amino acid elements, such as polyethylene glycols, lipids, poly- ormono-saccharide, and phosphates. Effects of such non-amino acid elementson the functionality of a ActRIIa polypeptide may be tested as describedherein for other ActRIIa polypeptide variants. When an ActRIIapolypeptide is produced in cells by cleaving a nascent form of theActRIIa polypeptide, post-translational processing may also be importantfor correct folding and/or function of the protein. Different cells(such as CHO, HeLa, MDCK, 293, WI38, NIH-3T3 or HEK293) have specificcellular machinery and characteristic mechanisms for suchpost-translational activities and may be chosen to ensure the correctmodification and processing of the ActRIIa polypeptides.

In certain aspects, functional variants or modified forms of the ActRIIapolypeptides include fusion proteins having at least a portion of theActRIIa polypeptides and one or more fusion domains. Well known examplesof such fusion domains include, but are not limited to, polyhistidine,Glu-Glu, glutathione S transferase (GST), thioredoxin, protein A,protein G, an immunoglobulin heavy chain constant region (Fc), maltosebinding protein (MBP), or human serum albumin. A fusion domain may beselected so as to confer a desired property. For example, some fusiondomains are particularly useful for isolation of the fusion proteins byaffinity chromatography. For the purpose of affinity purification,relevant matrices for affinity chromatography, such as glutathione-,amylase-, and nickel- or cobalt-conjugated resins are used. Many of suchmatrices are available in “kit” form, such as the Pharmacia GSTpurification system and the QIAexpress™ system (Qiagen) useful with(HIS₆) fusion partners. As another example, a fusion domain may beselected so as to facilitate detection of the ActRIIa polypeptides.Examples of such detection domains include the various fluorescentproteins (e.g., GFP) as well as “epitope tags,” which are usually shortpeptide sequences for which a specific antibody is available. Well knownepitope tags for which specific monoclonal antibodies are readilyavailable include FLAG, influenza virus haemagglutinin (HA), and c-myctags. In some cases, the fusion domains have a protease cleavage site,such as for Factor Xa or Thrombin, which allows the relevant protease topartially digest the fusion proteins and thereby liberate therecombinant proteins therefrom. The liberated proteins can then beisolated from the fusion domain by subsequent chromatographicseparation. In certain preferred embodiments, an ActRIIa polypeptide isfused with a domain that stabilizes the ActRIIa polypeptide in vivo (a“stabilizer” domain). By “stabilizing” is meant anything that increasesserum half life, regardless of whether this is because of decreaseddestruction, decreased clearance by the kidney, or other pharmacokineticeffect. Fusions with the Fc portion of an immunoglobulin are known toconfer desirable pharmacokinetic properties on a wide range of proteins.Likewise, fusions to human serum albumin can confer desirableproperties. Other types of fusion domains that may be selected includemultimerizing (e.g., dimerizing, tetramerizing) domains and functionaldomains (that confer an additional biological function, such as furtherstimulation of bone growth or muscle growth, as desired).

As a specific example, the present invention provides a fusion proteincomprising a soluble extracellular domain of ActRIIa fused to an Fcdomain (e.g., SEQ ID NO: 6).

THTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVD(A)VSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCK(A)VSNKALPVPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGPFFLYSKLTVDKSRWQQGNVFSCSVMHEALHN(A)HYTQKSLSLSPGK*

Optionally, the Fc domain has one or more mutations at residues such asAsp-265, lysine 322, and Asn-434. In certain cases, the mutant Fc domainhaving one or more of these mutations (e.g., Asp-265 mutation) hasreduced ability of binding to the Fcγ receptor relative to a wildtype Fcdomain. In other cases, the mutant Fc domain having one or more of thesemutations (e.g., Asn-434 mutation) has increased ability of binding tothe MHC class I-related Fc-receptor (FcRN) relative to a wildtype Fcdomain.

It is understood that different elements of the fusion proteins may bearranged in any manner that is consistent with the desiredfunctionality. For example, an ActRIIa polypeptide may be placedC-terminal to a heterologous domain, or, alternatively, a heterologousdomain may be placed C-terminal to an ActRIIa polypeptide. The ActRIIapolypeptide domain and the heterologous domain need not be adjacent in afusion protein, and additional domains or amino acid sequences may beincluded C- or N-terminal to either domain or between the domains.

In certain embodiments, the ActRIIa polypeptides of the presentinvention contain one or more modifications that are capable ofstabilizing the ActRIIa polypeptides. For example, such modificationsenhance the in vitro half life of the ActRIIa polypeptides, enhancecirculatory half life of the ActRIIa polypeptides or reduce proteolyticdegradation of the ActRIIa polypeptides. Such stabilizing modificationsinclude, but are not limited to, fusion proteins (including, forexample, fusion proteins comprising an ActRIIa polypeptide and astabilizer domain), modifications of a glycosylation site (including,for example, addition of a glycosylation site to an ActRIIapolypeptide), and modifications of carbohydrate moiety (including, forexample, removal of carbohydrate moieties from an ActRIIa polypeptide).In the case of fusion proteins, an ActRIIa polypeptide is fused to astabilizer domain such as an IgG molecule (e.g., an Fc domain). As usedherein, the term “stabilizer domain” not only refers to a fusion domain(e.g., Fc) as in the case of fusion proteins, but also includesnonproteinaceous modifications such as a carbohydrate moiety, ornonproteinaceous polymer, such as polyethylene glycol.

In certain embodiments, the present invention makes available isolatedand/or purified forms of the ActRIIa polypeptides, which are isolatedfrom, or otherwise substantially free of, other proteins. ActRIIapolypeptides will generally be produced by expression from recombinantnucleic acids.

3. Nucleic Acids Encoding ActRIIa Polypeptides

In certain aspects, the invention provides isolated and/or recombinantnucleic acids encoding any of the ActRIIa polypeptides (e.g., solubleActRIIa polypeptides), including fragments, functional variants andfusion proteins disclosed herein, and the use of nucleic acids toproduce protein for use in decreasing FSH levels. For example, SEQ IDNO: 4 encodes the naturally occurring human ActRIIa precursorpolypeptide, while SEQ ID NO: 5 encodes the processed extracellulardomain of ActRIIa. The subject nucleic acids may be single-stranded ordouble stranded. Such nucleic acids may be DNA or RNA molecules. Thesenucleic acids may be used, for example, in methods for making ActRIIapolypeptides or as direct therapeutic agents (e.g., in a gene therapyapproach).

In certain aspects, the subject nucleic acids encoding ActRIIapolypeptides are further understood to include nucleic acids that arevariants of SEQ ID NO: 4 or 5. Variant nucleotide sequences includesequences that differ by one or more nucleotide substitutions, additionsor deletions, such as allelic variants.

In certain embodiments, the invention provides for the use of isolatedor recombinant nucleic acid sequences that are at least 80%, 85%, 90%,95%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 4 or 5. One ofordinary skill in the art will appreciate that nucleic acid sequencescomplementary to SEQ ID NO: 4 or 5, and variants of SEQ ID NO: 4 or 5are also within the scope of this invention. In further embodiments, thenucleic acid sequences of the invention can be isolated, recombinant,and/or fused with a heterologous nucleotide sequence, or in a DNAlibrary.

In other embodiments, proteins to be used to decrease FSH levels areencoded by nucleic acids that hybridize under highly stringentconditions to the nucleotide sequence designated in SEQ ID NO: 4 or 5,complement sequence of SEQ ID NO: 4 or 5, or fragments thereof. Asdiscussed above, one of ordinary skill in the art will understandreadily that appropriate stringency conditions which promote DNAhybridization can be varied. One of ordinary skill in the art willunderstand readily that appropriate stringency conditions which promoteDNA hybridization can be varied. For example, one could perform thehybridization at 6.0× sodium chloride/sodium citrate (SSC) at about 45°C., followed by a wash of 2.0×SSC at 50° C. For example, the saltconcentration in the wash step can be selected from a low stringency ofabout 2.0×SSC at 50° C. to a high stringency of about 0.2×SSC at 50° C.In addition, the temperature in the wash step can be increased from lowstringency conditions at room temperature, about 22° C., to highstringency conditions at about 65° C. Both temperature and salt may bevaried, or temperature or salt concentration may be held constant whilethe other variable is changed. In one embodiment, the invention providesnucleic acids which hybridize under low stringency conditions of 6×SSCat room temperature followed by a wash at 2×SSC at room temperature.

Isolated nucleic acids which differ from the nucleic acids as set forthin SEQ ID NOs: 4 or 5 due to degeneracy in the genetic code are alsowithin the scope of the invention. For example, a number of amino acidsare designated by more than one triplet. Codons that specify the sameamino acid, or synonyms (for example, CAU and CAC are synonyms forhistidine) may result in “silent” mutations which do not affect theamino acid sequence of the protein. However, it is expected that DNAsequence polymorphisms that do lead to changes in the amino acidsequences of the subject proteins will exist among mammalian cells. Oneskilled in the art will appreciate that these variations in one or morenucleotides (up to about 3-5% of the nucleotides) of the nucleic acidsencoding a particular protein may exist among individuals of a givenspecies due to natural allelic variation. Any and all such nucleotidevariations and resulting amino acid polymorphisms are within the scopeof this invention.

In certain embodiments, the recombinant nucleic acids of the inventionmay be operably linked to one or more regulatory nucleotide sequences inan expression construct. Regulatory nucleotide sequences will generallybe appropriate to the host cell used for expression. Numerous types ofappropriate expression vectors and suitable regulatory sequences areknown in the art for a variety of host cells. Typically, said one ormore regulatory nucleotide sequences may include, but are not limitedto, promoter sequences, leader or signal sequences, ribosomal bindingsites, transcriptional start and termination sequences, translationalstart and termination sequences, and enhancer or activator sequences.Constitutive or inducible promoters as known in the art are contemplatedby the invention. The promoters may be either naturally occurringpromoters, or hybrid promoters that combine elements of more than onepromoter. An expression construct may be present in a cell on anepisome, such as a plasmid, or the expression construct may be insertedin a chromosome. In a preferred embodiment, the expression vectorcontains a selectable marker gene to allow the selection of transformedhost cells. Selectable marker genes are well known in the art and willvary with the host cell used.

In certain aspects of the invention, the subject nucleic acid isprovided in an expression vector comprising a nucleotide sequenceencoding an ActRIIa polypeptide and operably linked to at least oneregulatory sequence. Regulatory sequences are art-recognized and areselected to direct expression of the ActRIIa polypeptide. Accordingly,the term regulatory sequence includes promoters, enhancers, and otherexpression control elements. Exemplary regulatory sequences aredescribed in Goeddel; Gene Expression Technology: Methods in Enzymology,Academic Press, San Diego, Calif. (1990). For instance, any of a widevariety of expression control sequences that control the expression of aDNA sequence when operatively linked to it may be used in these vectorsto express DNA sequences encoding an ActRIIa polypeptide. Such usefulexpression control sequences, include, for example, the early and latepromoters of SV40, tet promoter, adenovirus or cytomegalovirus immediateearly promoter, RSV promoters, the lac system, the trp system, the TACor TRC system, T7 promoter whose expression is directed by T7 RNApolymerase, the major operator and promoter regions of phage lambda, thecontrol regions for fd coat protein, the promoter for 3-phosphoglyceratekinase or other glycolytic enzymes, the promoters of acid phosphatase,e.g., Pho5, the promoters of the yeast α-mating factors, the polyhedronpromoter of the baculovirus system and other sequences known to controlthe expression of genes of prokaryotic or eukaryotic cells or theirviruses, and various combinations thereof. It should be understood thatthe design of the expression vector may depend on such factors as thechoice of the host cell to be transformed and/or the type of proteindesired to be expressed. Moreover, the vector's copy number, the abilityto control that copy number and the expression of any other proteinencoded by the vector, such as antibiotic markers, should also beconsidered.

A recombinant nucleic acid of the invention can be produced by ligatingthe cloned gene, or a portion thereof, into a vector suitable forexpression in either prokaryotic cells, eukaryotic cells (yeast, avian,insect or mammalian), or both. Expression vehicles for production of arecombinant ActRIIa polypeptide include plasmids and other vectors. Forinstance, suitable vectors include plasmids of the types: pBR322-derivedplasmids, pEMBL-derived plasmids, pEX-derived plasmids, pBTac-derivedplasmids and pUC-derived plasmids for expression in prokaryotic cells,such as E. coli.

Some mammalian expression vectors contain both prokaryotic sequences tofacilitate the propagation of the vector in bacteria, and one or moreeukaryotic transcription units that are expressed in eukaryotic cells.The pcDNAI/amp, pcDNAI/neo, pRc/CMV, pSV2gpt, pSV2neo, pSV2-dhfr, pTk2,pRSVneo, pMSG, pSVT7, pko-neo and pHyg derived vectors are examples ofmammalian expression vectors suitable for transfection of eukaryoticcells. Some of these vectors are modified with sequences from bacterialplasmids, such as pBR322, to facilitate replication and drug resistanceselection in both prokaryotic and eukaryotic cells. Alternatively,derivatives of viruses such as the bovine papilloma virus (BPV-1), orEpstein-Barr virus (pHEBo, pREP-derived and p205) can be used fortransient expression of proteins in eukaryotic cells. Examples of otherviral (including retroviral) expression systems can be found below inthe description of gene therapy delivery systems. The various methodsemployed in the preparation of the plasmids and in transformation ofhost organisms are well known in the art. For other suitable expressionsystems for both prokaryotic and eukaryotic cells, as well as generalrecombinant procedures, see Molecular Cloning A Laboratory Manual, 3rdEd., ed. by Sambrook, Fritsch and Maniatis (Cold Spring HarborLaboratory Press, 2001). In some instances, it may be desirable toexpress the recombinant polypeptides by the use of a baculovirusexpression system. Examples of such baculovirus expression systemsinclude pVL-derived vectors (such as pVL1392, pVL1393 and pVL941),pAcUW-derived vectors (such as pAcUW1), and pBlueBac-derived vectors(such as the β-gal containing pBlueBac III).

In a preferred embodiment, a vector will be designed for production ofthe subject ActRIIa polypeptides in CHO cells, such as a Pcmv-Scriptvector (Stratagene, La Jolla, Calif), pcDNA4 vectors (Invitrogen,Carlsbad, Calif.) and pCI-neo vectors (Promega, Madison, Wis.). As willbe apparent, the subject gene constructs can be used to cause expressionof the subject ActRIIa polypeptides in cells propagated in culture,e.g., to produce proteins, including fusion proteins or variantproteins, for purification.

This disclosure also pertains to a host cell transfected with arecombinant gene including a coding sequence (e.g., SEQ ID NO: 4 or 5)for one or more of the subject ActRIIa polypeptides. The host cell maybe any prokaryotic or eukaryotic cell. For example, an ActRIIapolypeptide of the invention may be expressed in bacterial cells such asE. coli, insect cells (e.g., using a baculovirus expression system),yeast, or mammalian cells. Other suitable host cells are known to thoseskilled in the art.

Accordingly, the present invention further pertains to methods ofproducing the subject ActRIIa polypeptides. For example, a host celltransfected with an expression vector encoding an ActRIIa polypeptidecan be cultured under appropriate conditions to allow expression of theActRIIa polypeptide to occur. The ActRIIa polypeptide may be secretedand isolated from a mixture of cells and medium containing the ActRIIapolypeptide. Alternatively, the ActRIIa polypeptide may be retainedcytoplasmically or in a membrane fraction and the cells harvested, lysedand the protein isolated. A cell culture includes host cells, media andother byproducts. Suitable media for cell culture are well known in theart. The subject ActRIIa polypeptides can be isolated from cell culturemedium, host cells, or both, using techniques known in the art forpurifying proteins, including ion-exchange chromatography, gelfiltration chromatography, ultrafiltration, electrophoresis,immunoaffinity purification with antibodies specific for particularepitopes of the ActRIIa polypeptides and affinity purification with anagent that binds to a domain fused to the ActRIIa polypeptide (e.g., aprotein A column may be used to purify an ActRIIa-Fc fusion). In apreferred embodiment, the ActRIIa polypeptide is a fusion proteincontaining a domain which facilitates its purification. In a preferredembodiment, purification is achieved by a series of columnchromatography steps, including, for example, three or more of thefollowing, in any order: protein A chromatography, Q sepharosechromatography, phenylsepharose chromatography, size exclusionchromatography, and cation exchange chromatography. The purificationcould be completed with viral filtration and buffer exchange. Asdemonstrated herein, ActRIIa-hFc protein was purified to a purityof >98% as determined by size exclusion chromatography and >95% asdetermined by SDS PAGE. This level of purity was sufficient to achievedesirable effects on bone in mice and an acceptable safety profile inmice, rats and non-human primates.

In another embodiment, a fusion gene coding for a purification leadersequence, such as a poly-(His)/enterokinase cleavage site sequence atthe N-terminus of the desired portion of the recombinant ActRIIapolypeptide, can allow purification of the expressed fusion protein byaffinity chromatography using a Ni²⁺ metal resin. The purificationleader sequence can then be subsequently removed by treatment withenterokinase to provide the purified ActRIIa polypeptide (e.g., seeHochuli et al., (1987) J. Chromatography 411:177; and Janknecht et al.,PNAS USA 88:8972).

Techniques for making fusion genes are well known. Essentially, thejoining of various DNA fragments coding for different polypeptidesequences is performed in accordance with conventional techniques,employing blunt-ended or stagger-ended termini for ligation, restrictionenzyme digestion to provide for appropriate termini, filling-in ofcohesive ends as appropriate, alkaline phosphatase treatment to avoidundesirable joining, and enzymatic ligation. In another embodiment, thefusion gene can be synthesized by conventional techniques includingautomated DNA synthesizers. Alternatively, PCR amplification of genefragments can be carried out using anchor primers which give rise tocomplementary overhangs between two consecutive gene fragments which cansubsequently be annealed to generate a chimeric gene sequence (see, forexample, Current Protocols in Molecular Biology, eds. Ausubel et al.,John Wiley & Sons: 1992).

4. Alternative Activin and ActRIIa Antagonists

The data presented herein demonstrates that antagonists ofactivin-ActRIIa signaling can be used to decrease FSH levels. Althoughsoluble ActRIIa polypeptides, and particularly ActrIIa-Fc, are preferredantagonists, and although such antagonists may affect FSH through amechanism other than activin antagonism, other types of activin-ActRIIaantagonists are expected to be useful, including anti-activin (e.g., A,B, C or E) antibodies, anti-ActRIIa antibodies, antisense, RNAi orribozyme nucleic acids that inhibit the production of ActRIIa and otherinhibitors of activin or ActRIIa, particularly those that disruptactivin-ActRIIa binding.

An antibody that is specifically reactive with an ActRIIa polypeptide(e.g., a soluble ActRIIa polypeptide) and which either bindscompetitively to ligand with the ActRIIa polypeptide or otherwiseinhibits ActRIIa-mediated signaling may be used as an antagonist ofActRIIa polypeptide activities. Likewise, an antibody that isspecifically reactive with an activin A polypeptide and which disruptsActRIIa binding may be used as an antagonist.

By using immunogens derived from an ActRIIa polypeptide or an activinpolypeptide, anti-protein/anti-peptide antisera or monoclonal antibodiescan be made by standard protocols (see, for example, Antibodies: ALaboratory Manual ed. by Harlow and Lane (Cold Spring Harbor Press:1988)). A mammal, such as a mouse, a hamster or rabbit can be immunizedwith an immunogenic form of the ActRIIa polypeptide, an antigenicfragment which is capable of eliciting an antibody response, or a fusionprotein. Techniques for conferring immunogenicity on a protein orpeptide include conjugation to carriers or other techniques well knownin the art. An immunogenic portion of an ActRIIa or activin polypeptidecan be administered in the presence of adjuvant. The progress ofimmunization can be monitored by detection of antibody titers in plasmaor serum. Standard ELISA or other immunoassays can be used with theimmunogen as antigen to assess the levels of antibodies.

Following immunization of an animal with an antigenic preparation of anActRIIa polypeptide, antisera can be obtained and, if desired,polyclonal antibodies can be isolated from the serum. To producemonoclonal antibodies, antibody-producing cells (lymphocytes) can beharvested from an immunized animal and fused by standard somatic cellfusion procedures with immortalizing cells such as myeloma cells toyield hybridoma cells. Such techniques are well known in the art, andinclude, for example, the hybridoma technique (originally developed byKohler and Milstein, (1975) Nature, 256: 495-497), the human B cellhybridoma technique (Kozbar et al., (1983) Immunology Today, 4: 72), andthe EBV-hybridoma technique to produce human monoclonal antibodies (Coleet al., (1985) Monoclonal Antibodies and Cancer Therapy, Alan R. Liss,Inc. pp. 77-96). Hybridoma cells can be screened immunochemically forproduction of antibodies specifically reactive with an ActRIIapolypeptide and monoclonal antibodies isolated from a culture comprisingsuch hybridoma cells.

The term “antibody” as used herein is intended to include fragmentsthereof which are also specifically reactive with a subject polypeptide.Antibodies can be fragmented using conventional techniques and thefragments screened for utility in the same manner as described above forwhole antibodies. For example, F(ab)₂ fragments can be generated bytreating antibody with pepsin. The resulting F(ab)₂ fragment can betreated to reduce disulfide bridges to produce Fab fragments. Theantibody of the present invention is further intended to includebispecific, single-chain, chimeric, humanized and fully human moleculeshaving affinity for an ActRIIa or activin polypeptide conferred by atleast one CDR region of the antibody. An antibody may further comprise alabel attached thereto and able to be detected (e.g., the label can be aradioisotope, fluorescent compound, enzyme or enzyme co-factor).

In certain embodiments, the antibody is a recombinant antibody, whichterm encompasses any antibody generated in part by techniques ofmolecular biology, including CDR-grafted or chimeric antibodies, humanor other antibodies assembled from library-selected antibody domains,single chain antibodies and single domain antibodies (e.g., human V_(H)proteins or camelid V_(HH) proteins). In certain embodiments, anantibody of the invention is a monoclonal antibody, and in certainembodiments, the invention makes available methods for generating novelantibodies. For example, a method for generating a monoclonal antibodythat binds specifically to an ActRIIa polypeptide or activin polypeptidemay comprise administering to a mouse an amount of an immunogeniccomposition comprising the antigen polypeptide effective to stimulate adetectable immune response, obtaining antibody-producing cells (e.g.,cells from the spleen) from the mouse and fusing the antibody-producingcells with myeloma cells to obtain antibody-producing hybridomas, andtesting the antibody-producing hybridomas to identify a hybridoma thatproduces a monocolonal antibody that binds specifically to the antigen.Once obtained, a hybridoma can be propagated in a cell culture,optionally in culture conditions where the hybridoma-derived cellsproduce the monoclonal antibody that binds specifically to the antigen.The monoclonal antibody may be purified from the cell culture.

The adjective “specifically reactive with” as used in reference to anantibody is intended to mean, as is generally understood in the art,that the antibody is sufficiently selective between the antigen ofinterest (e.g., an ActRIIa polypeptide) and other antigens that are notof interest that the antibody is useful for, at minimum, detecting thepresence of the antigen of interest in a particular type of biologicalsample. In certain methods employing the antibody, such as therapeuticapplications, a higher degree of specificity in binding may bedesirable. Monoclonal antibodies generally have a greater tendency (ascompared to polyclonal antibodies) to discriminate effectively betweenthe desired antigens and cross-reacting polypeptides. One characteristicthat influences the specificity of an antibody:antigen interaction isthe affinity of the antibody for the antigen. Although the desiredspecificity may be reached with a range of different affinities,generally preferred antibodies will have an affinity (a dissociationconstant) of about 10⁻⁶, 10⁻⁷, 10⁻⁸, 10⁻⁹ or less. Given theextraordinarily tight binding between activin and ActRIIa, it isexpected that a neutralizing anti-activin or anti-ActRIIa antibody wouldgenerally have a dissociation constant of 10⁻¹⁰ or less.

In addition, the techniques used to screen antibodies in order toidentify a desirable antibody may influence the properties of theantibody obtained. For example, if an antibody is to be used for bindingan antigen in solution, it may be desirable to test solution binding. Avariety of different techniques are available for testing interactionbetween antibodies and antigens to identify particularly desirableantibodies. Such techniques include ELISAs, surface plasmon resonancebinding assays (e.g., the Biacore™ binding assay, Biacore AB, Uppsala,Sweden), sandwich assays (e.g., the paramagnetic bead system of IGENInternational, Inc., Gaithersburg, Md.), western blots,immunoprecipitation assays, and immunohistochemistry.

Examples of categories of nucleic acid compounds that are activin orActRIIa antagonists include antisense nucleic acids, RNAi constructs andcatalytic nucleic acid constructs. A nucleic acid compound may be singleor double stranded. A double stranded compound may also include regionsof overhang or non-complementarity, where one or the other of thestrands is single stranded. A single stranded compound may includeregions of self-complementarity, meaning that the compound forms aso-called “hairpin” or “stem-loop” structure, with a region of doublehelical structure. A nucleic acid compound may comprise a nucleotidesequence that is complementary to a region consisting of no more than1000, no more than 500, no more than 250, no more than 100 or no morethan 50, 35, 30, 25, 22, 20 or 18 nucleotides of the full-length ActRIIanucleic acid sequence or activin βA or activin βB nucleic acid sequence.The region of complementarity will preferably be at least 8 nucleotides,and optionally at least 10 or at least 15 nucleotides, and optionallybetween 15 and 25 nucleotides. A region of complementarity may fallwithin an intron, a coding sequence or a noncoding sequence of thetarget transcript, such as the coding sequence portion. Generally, anucleic acid compound will have a length of about 8 to about 500nucleotides or base pairs in length, and optionally the length will beabout 14 to about 50 nucleotides. A nucleic acid may be a DNA(particularly for use as an antisense), RNA or RNA:DNA hybrid. Any onestrand may include a mixture of DNA and RNA, as well as modified formsthat cannot readily be classified as either DNA or RNA. Likewise, adouble stranded compound may be DNA:DNA, DNA:RNA or RNA:RNA, and any onestrand may also include a mixture of DNA and RNA, as well as modifiedforms that cannot readily be classified as either DNA or RNA. A nucleicacid compound may include any of a variety of modifications, includingone or modifications to the backbone (the sugar-phosphate portion in anatural nucleic acid, including internucleotide linkages) or the baseportion (the purine or pyrimidine portion of a natural nucleic acid). Anantisense nucleic acid compound will preferably have a length of about15 to about 30 nucleotides and will often contain one or moremodifications to improve characteristics such as stability in the serum,in a cell or in a place where the compound is likely to be delivered,such as the stomach in the case of orally delivered compounds and thelung for inhaled compounds. In the case of an RNAi construct, the strandcomplementary to the target transcript will generally be RNA ormodifications thereof. The other strand may be RNA, DNA or any othervariation. The duplex portion of double stranded or single stranded“hairpin” RNAi construct will preferably have a length of 18 to 40nucleotides in length and optionally about 21 to 23 nucleotides inlength, so long as it serves as a Dicer substrate. Catalytic orenzymatic nucleic acids may be ribozymes or DNA enzymes and may alsocontain modified forms. Nucleic acid compounds may inhibit expression ofthe target by about 50%, 75%, 90% or more when contacted with cellsunder physiological conditions and at a concentration where a nonsenseor sense control has little or no effect. Preferred concentrations fortesting the effect of nucleic acid compounds are 1, 5 and 10 micromolar.Nucleic acid compounds may also be tested for effects on, for example,FSH levels in vivo, FSH production by cell lines in vitro, orFSH-related disorders.

5. Screening Assays

In certain aspects, the present invention relates to the use of ActRIIapolypeptides (e.g., soluble ActRIIa polypeptides) and activinpolypeptides to identify compounds (agents) which are agonist orantagonists of the activin-ActRIIa signaling pathway. Compoundsidentified through this screening can be tested to assess their abilityto modulate the growth or survival of cancer cells, particularlyprostate cancer cells, in vivo or in vitro. These compounds can betested, for example, in animal models such as mouse xenograft models.One useful animal model is the murine LAPC-4 prostate cancer model(described in U.S. Pat. No. 7,122,714). Other animal models of prostatecancer can be generated, for example, by implanting LNCaP cells. TheLNCaP cell line is an established androgen-responsive prostate cancercell line obtained from a lymph node metastasis of a prostate cancerpatient.

There are numerous approaches to screening for therapeutic agents fordecreasing or inhibiting FSH secretion by targeting activin and ActRIIasignaling. In certain embodiments, high-throughput screening ofcompounds can be carried out to identify agents that perturb activin orActRIIa-mediated effects on a selected cell line. In certainembodiments, the assay is carried out to screen and identify compoundsthat specifically inhibit or reduce binding of an ActRIIa polypeptide toactivin. Alternatively, the assay can be used to identify compounds thatenhance binding of an ActRIIa polypeptide to activin. In a furtherembodiment, the compounds can be identified by their ability to interactwith an activin or ActRIIa polypeptide.

A variety of assay formats will suffice and, in light of the presentdisclosure, those not expressly described herein will nevertheless becomprehended by one of ordinary skill in the art. As described herein,test compounds (agents) may be created by any combinatorial chemicalmethod. Alternatively, the subject compounds may be naturally occurringbiomolecules synthesized in vivo or in vitro. Compounds (agents) to betested for their ability to act as modulators of tissue growth can beproduced, for example, by bacteria, yeast, plants or other organisms(e.g., natural products), produced chemically (e.g., small molecules,including peptidomimetics), or produced recombinantly. Test compoundscontemplated herein include non-peptidyl organic molecules, peptides,polypeptides, peptidomimetics, sugars, hormones, and nucleic acidmolecules. In a specific embodiment, the test agent is a small organicmolecule having a molecular weight of less than about 2,000 Daltons.

Test compounds can be provided as single, discrete entities, or providedin libraries of greater complexity, such as made by combinatorialchemistry. These libraries can comprise, for example, alcohols, alkylhalides, amines, amides, esters, aldehydes, ethers and other classes oforganic compounds. Presentation of test compounds to the test system canbe in either an isolated form or as mixtures of compounds, especially ininitial screening steps. Optionally, the compounds may be optionallyderivatized with other compounds and have derivatizing groups thatfacilitate isolation of the compounds. Non-limiting examples ofderivatizing groups include biotin, fluorescein, digoxygenin, greenfluorescent protein, isotopes, polyhistidine, magnetic beads,glutathione S transferase (GST), photoactivatible crosslinkers or anycombinations thereof.

In many drug screening programs which test libraries of compounds andnatural extracts, high throughput assays are desirable in order tomaximize the number of compounds surveyed in a given period of time.Assays which are performed in cell-free systems, such as may be derivedwith purified or semi-purified proteins, are often preferred as“primary” screens in that they can be generated to permit rapiddevelopment and relatively easy detection of an alteration in amolecular target which is mediated by a test compound. Moreover, theeffects of cellular toxicity or bioavailability of the test compound canbe generally ignored in the in vitro system, the assay instead beingfocused primarily on the effect of the drug on the molecular target asmay be manifest in an alteration of binding affinity between an ActRIIapolypeptide and activin.

Merely to illustrate, in an exemplary screening assay, the compound ofinterest is contacted with an isolated and purified ActRIIa polypeptidewhich is ordinarily capable of binding to activin. To the mixture of thecompound and ActRIIa polypeptide is then added a composition containingan ActRIIa ligand. Detection and quantification of ActRIIa/activincomplexes provides a means for determining the compound's efficacy atinhibiting (or potentiating) complex formation between the ActRIIapolypeptide and activin. The efficacy of the compound can be assessed bygenerating dose response curves from data obtained using variousconcentrations of the test compound. Moreover, a control assay can alsobe performed to provide a baseline for comparison. For example, in acontrol assay, isolated and purified activin is added to a compositioncontaining the ActRIIa polypeptide, and the formation of ActRIIa/activincomplex is quantitated in the absence of the test compound. It will beunderstood that, in general, the order in which the reactants may beadmixed can be varied, and can be admixed simultaneously. Moreover, inplace of purified proteins, cellular extracts and lysates may be used torender a suitable cell-free assay system.

Complex formation between the ActRIIa polypeptide and activin may bedetected by a variety of techniques. For instance, modulation of theformation of complexes can be quantitated using, for example, detectablylabeled proteins such as radiolabeled (e.g., ³²P, ³⁵S, ¹⁴C or ³H),fluorescently labeled (e.g., FITC), or enzymatically labeled ActRIIapolypeptide or activin, by immunoassay, or by chromatographic detection.

In certain embodiments, fluorescence polarization assays andfluorescence resonance energy transfer (FRET) assays may be used formeasuring, either directly or indirectly, the degree of interactionbetween an ActRIIa polypeptide and its binding protein. Other suitablemodes of detection include, for example, those based on opticalwaveguides (PCT Publication WO 96/26432 and U.S. Pat. No. 5,677,196),surface plasmon resonance (SPR), surface charge sensors, and surfaceforce sensors.

An interaction trap assay, also known as the “two hybrid assay,” mayalso be used for identifying agents that disrupt or potentiateinteraction between an ActRIIa polypeptide and its binding protein. Seefor example, U.S. Pat. No. 5,283,317; Zervos et al. (1993) Cell72:223-232; Madura et al. (1993) J Biol Chem 268:12046-12054; Bartel etal. (1993) Biotechniques 14:920-924; and Iwabuchi et al. (1993) Oncogene8:1693-1696). In a specific embodiment, a reverse two hybrid system maybe used to identify compounds (e.g., small molecules or peptides) thatdissociate interactions between an ActRIIa polypeptide and its bindingprotein. See for example, Vidal and Legrain, (1999) Nucleic Acids Res27:919-29; Vidal and Legrain, (1999) Trends Biotechnol 17:374-81; andU.S. Pat. Nos. 5,525,490; 5,955,280; and 5,965,368.

In certain embodiments, compounds are identified by their ability tointeract with an ActRIIa or activin polypeptide described herein. Theinteraction between the compound and the ActRIIa or activin polypeptidemay be covalent or non-covalent. For example, such interaction can beidentified at the protein level using in vitro biochemical methods,including photo-crosslinking, radiolabeled ligand binding, and affinitychromatography (Jakoby W B et al., 1974, Methods in Enzymology 46: 1).In certain cases, the compounds may be screened in a mechanism basedassay, such as an assay to detect compounds which bind to an activin orActRIIa polypeptide. This may include a solid phase or fluid phasebinding event. Alternatively, the gene encoding an activin or ActRIIapolypeptide can be transfected with a reporter system (e.g.,β-galactosidase, luciferase, or green fluorescent protein) into a celland screened against the library optionally by a high throughputscreening or with individual members of the library. Other mechanismbased binding assays may be used, for example, binding assays whichdetect changes in free energy. Binding assays can be performed with thetarget fixed to a well, bead or chip or captured by an immobilizedantibody or resolved by capillary electrophoresis. The bound compoundsmay be detected usually using colorimetric or fluorescence or surfaceplasmon resonance.

6. Exemplary Therapeutic Uses

In certain embodiments, the present invention provides methods ofdecreasing or inhibiting FSH secretion in an individual in need thereofby administering to the individual a therapeutically effective amount ofan activin-ActRIIa antagonist, such as, for example, an ActRIIapolypeptide. Methods of decreasing or inhibiting FSH secretion includeall methods which lead to said effect, including, for example,decreasing FSH transcription, translation, post-translationalprocessing, and secretion. Various kits are available for testing plasmaFSH levels, including MENOCHECK™. Normal values for FSH in men rangefrom 2-18 mIU/ml of blood. Normal values for women range from 5 and 25mIU/mL. Levels higher than 50 mIU/mL in healthy women are associatedwith menopause. The tissue concentration of FSH can be determined bytesting saliva (eMHP™).

In certain embodiments, the present invention provides methods oftreating or preventing prostate cancer in an individual in need thereofby administering to the individual a therapeutically effective amount ofan activin-ActRIIa antagonist, such as, for example, an ActRIIapolypeptide in order to decrease or inhibit FSH secretion. These methodsmay be used for therapeutic as well as prophylactic treatment of humans,particularly males, who have a high risk for developing prostate cancer.As every man is at risk for developing prostate cancer, a man with ahigh risk for developing prostate cancer is a man whose risk factorsconfer a greater probability of developing the disease compared to thegeneral population or the population of men within a certain age group.Exemplary risk factors include age, family history or genetic makeup,lifestyle habits such as exercise and diet, and exposure to radiation orother cancer-causing agents.

As used herein, a therapeutic that “prevents” a disorder or conditionrefers to a compound that, in a statistical sample, reduces theoccurrence of the disorder or condition in the treated sample relativeto an untreated control sample, or delays the onset of one or moresymptoms or characteristics of the disorder or condition relative to theuntreated control sample. For example, preventing prostate cancer mayrefer to the absence of new lesions following treatment, or the absenceor delay of metastatic disease.

The term “treating prostate cancer” refers to an improvement of one ormore symptoms or characteristics of the disease relative to an untreatedcontrol or relative to the severity of disease prior to treatment. Theterm does not necessarily require that the patient receiving thetreatment be cured or that the disease be completely eradicated from thepatient. An agent that treats prostate cancer may be an agent thatreduces the severity of one or more symptoms or characteristics of thedisease. It should be noted that tumor growth and progression isinfluenced by a variety of factors, including mediators of cell cycleprogression and cell division and regulators of cell death, orapoptosis. Accordingly, treating prostate cancer may involve a decreasein cancer cell proliferation or a decrease in the rate of cell division.Alternatively or additionally, treating prostate cancer may involve adecrease in cancer cell survival or an increase in apoptosis.Accordingly, in certain embodiments, treating prostate cancer mayinvolve both a decrease in cell division and an increase in cell death.Regardless of mechanism, the effectiveness of an agent in treatingprostate cancer may be determined by observable metrics, such as a lowernumber of cancer cells compared to a control (either due to decreasedproliferation, increased apoptosis, or both), or a decrease in tumorsize compared to a control. Therefore treating prostate cancer orinhibiting tumor or cancer cell growth is intended to be neutral as tothe mechanism by which such a change occurs. Both prevention andtreatment may be discerned in the diagnosis provided by a physician orother health care provider and the analysis of the intended result ofadministration of the therapeutic agent.

When observing the effects of the subject antagonists on prostate cancerprogression in humans, an effect may be evaluated by a decrease ordisappearance of measurable disease, and/or the absence of new lesionsor the prevention of metastases. For example, activin-ActRIIaantagonists may significantly reduce or delay prostate cancerprogression in patients with both noninvasive and invasive prostatecancer. In addition, the antagonists may prevent or reduce the risk ofdeveloping prostate cancer in healthy men with risk factors for thedisease. The antagonists may also reduce the risk of prostate cancerrecurrence in patients with a history of the disease.

Accordingly, activin-ActRIIa antagonists may be used to prevent or delaythe onset of prostate cancer in individuals considered to be at risk fordeveloping the disease, and such antagonists may be used in selectedpatient populations. Examples of appropriate patient populations includepatients with a family history of prostate cancer, such as male patientswhere a father or brother has been diagnosed with the disease. In oneembodiment, a patient considered to be at high risk for developingprostate cancer but who has not been diagnosed with the disease istreated with an activin-ActRIIa antagonist. Such treatment may beginwhen the patient reaches the age of 30, 40, 50, 60, or 70.

Activin-ActRIIa antagonists disclosed herein, and particularlyActRIIa-Fc proteins, may be used to treat or prevent prostate cancer ina patient, including patients with solid tumors as well as patients withmetastatic cancer. Activin-ActRIIa antagonists may also be administeredto human subjects with precancerous or benign lesions of the prostate orwith any abnormal proliferative lesions including typical hyperplasia,atypical hyperplasia, and noninvasive or in situ carcinoma. Theantagonists of the present disclosure are also useful in the treatmentor prevention of both hormone-dependent or hormone-responsive cancersand hormone-independent cancers (e.g., hormone-refractory prostatecancer). Activin-ActRIIa antagonists may prove to be particularly usefulin tumors that express elevated (relative to normal prostatetissue-derived cells) levels of activin (e.g., A, AB or B) or elevatedlevels of ActRIIa or ActRIIb.

In certain embodiments, the present invention provides methods ofdecreasing or inhibiting FSH secretion in an individual afflicted withan FSH-secreting pituitary tumor by administering to the individual atherapeutically effective amount of an activin-ActRIIa antagonist, suchas, for example, an ActRIIa polypeptide. Inhibiting the hyper-secretionof FSH in these pituitary tumors is useful as a treatment to reduce thetumor symptoms, such as increased estrogen levels and the development ofovarian cysts. The present methods are preferably used in conjunctionwith conventional cancer therapies, such as surgery, however, theinhibition of FSH secretion alone may be an effective treatment,especially in cases where surgery or radiation is contraindicated.

The present invention recognizes that the effectiveness of conventionalcancer therapies (e.g., chemotherapy, radiation therapy, phototherapy,immunotherapy, and surgery, in particular prostatectomy) can be enhancedthrough the use of the subject antagonists. Accordingly, activin-ActRIIaantagonists may be used in combination therapies for the treatment,prevention, or management of prostate cancer. The antagonists may beadministered to patients in combination with radiation and/or surgicaltreatment as well as with cytotoxic chemotherapy and/or endocrinetherapies. Such combination treatments may work synergistically andallow reduction of dosage of each of the individual treatments, therebyreducing the detrimental side effects exerted by each treatment athigher dosages. In other instances, malignancies that are refractory toa treatment may respond to a combination therapy of two or moredifferent treatments. Accordingly, the disclosure relates to theadministration of an activin-ActRIIa antagonist in combination withanother conventional anti-neoplastic agent, either concomitantly orsequentially, in order to enhance the therapeutic effect of theanti-neoplastic agent or overcome cellular resistance to suchanti-neoplastic agent. The disclosure also relates to the administrationof an activin-ActRIIa antagonist in combination with hormonal therapy.Activin-ActRIIa antagonists may also be used in combination therapies toreduce the symptoms arising from FSH secreting pituitary tumors.Pharmaceutical compounds that may be used for combinatory anti-tumortherapy include, merely to illustrate: aminoglutethimide, amsacrine,anastrozole, asparaginase, bcg, bicalutamide, bleomycin, buserelin,busulfan, campothecin, capecitabine, carboplatin, carmustine,chlorambucil, cisplatin, cladribine, clodronate, colchicine,cyclophosphamide, cyproterone, cytarabine, dacarbazine, dactinomycin,daunorubicin, dienestrol, diethylstilbestrol, docetaxel, doxorubicin,epirubicin, estradiol, estramustine, etoposide, exemestane, filgrastim,fludarabine, fludrocortisone, fluorouracil, fluoxymesterone, flutamide,gemcitabine, genistein, goserelin, hydroxyurea, idarubicin, ifosfamide,imatinib, interferon, irinotecan, ironotecan, letrozole, leucovorin,leuprolide, levamisole, lomustine, mechlorethamine, medroxyprogesterone,megestrol, melphalan, mercaptopurine, mesna, methotrexate, mitomycin,mitotane, mitoxantrone, nilutamide, nocodazole, octreotide, oxaliplatin,paclitaxel, pamidronate, pentostatin, plicamycin, porfimer,procarbazine, raltitrexed, rituximab, streptozocin, suramin, tamoxifen,temozolomide, teniposide, testosterone, thioguanine, thiotepa,titanocene dichloride, topotecan, trastuzumab, tretinoin, vinblastine,vincristine, vindesine, and vinorelbine.

These chemotherapeutic anti-tumor compounds may be categorized by theirmechanism of action into, for example, following groups:anti-metabolites/anti-cancer agents, such as pyrimidine analogs(5-fluorouracil, floxuridine, capecitabine, gemcitabine and cytarabine)and purine analogs, folate antagonists and related inhibitors(mercaptopurine, thioguanine, pentostatin and 2-chlorodeoxyadenosine(cladribine)); antiproliferative/antimitotic agents including naturalproducts such as vinca alkaloids (vinblastine, vincristine, andvinorelbine), microtubule disruptors such as taxane (paclitaxel,docetaxel), vincristin, vinblastin, nocodazole, epothilones andnavelbine, epidipodophyllotoxins (etoposide, teniposide), DNA damagingagents (actinomycin, amsacrine, anthracyclines, bleomycin, busulfan,camptothecin, carboplatin, chlorambucil, cisplatin, cyclophosphamide,cytoxan, dactinomycin, daunorubicin, doxorubicin, epirubicin,hexamethylmelamineoxaliplatin, iphosphamide, melphalan,merchlorehtamine, mitomycin, mitoxantrone, nitrosourea, plicamycin,procarbazine, taxol, taxotere, teniposide, triethylenethiophosphoramideand etoposide (VP16)); antibiotics such as dactinomycin (actinomycin D),daunorubicin, doxorubicin (adriamycin), idarubicin, anthracyclines,mitoxantrone, bleomycins, plicamycin (mithramycin) and mitomycin;enzymes (L-asparaginase which systemically metabolizes L-asparagine anddeprives cells which do not have the capacity to synthesize their ownasparagine); antiplatelet agents; antiproliferative/antimitoticalkylating agents such as nitrogen mustards (mechlorethamine,cyclophosphamide and analogs, melphalan, chlorambucil), ethyleniminesand methylmelamines (hexamethylmelamine and thiotepa), alkylsulfonates-busulfan, nitrosoureas (carmustine (BCNU) and analogs,streptozocin), trazenes-dacarbazinine (DTIC);antiproliferative/antimitotic antimetabolites such as folic acid analogs(methotrexate); platinum coordination complexes (cisplatin,carboplatin), procarbazine, hydroxyurea, mitotane, aminoglutethimide;hormones, hormone analogs (estrogen, tamoxifen, goserelin, bicalutamide,nilutamide) and aromatase inhibitors (letrozole, anastrozole);anticoagulants (heparin, synthetic heparin salts and other inhibitors ofthrombin); fibrinolytic agents (such as tissue plasminogen activator,streptokinase and urokinase), aspirin, dipyridamole, ticlopidine,clopidogrel, abciximab; antimigratory agents; antisecretory agents(breveldin); immunosuppressives (cyclosporine, tacrolimus (FK-506),sirolimus (rapamycin), azathioprine, mycophenolate mofetil);anti-angiogenic compounds (TNP-470, genistein) and growth factorinhibitors (vascular endothelial growth factor (VEGF) inhibitors,fibroblast growth factor (FGF) inhibitors); angiotensin receptorblocker; nitric oxide donors; anti-sense oligonucleotides; antibodies(trastuzumab); cell cycle inhibitors and differentiation inducers(tretinoin); mTOR inhibitors, topoisomerase inhibitors (doxorubicin(adriamycin), amsacrine, camptothecin, daunorubicin, dactinomycin,eniposide, epirubicin, etoposide, idarubicin and mitoxantrone,topotecan, irinotecan), corticosteroids (cortisone, dexamethasone,hydrocortisone, methylpednisolone, prednisone, and prenisolone); growthfactor signal transduction kinase inhibitors; mitochondrial dysfunctioninducers and caspase activators; and chromatin disruptors.

In certain embodiments, pharmaceutical compounds that may be used forcombinatory therapy include anti-angiogenesis agents such as (1)inhibitors of release of “angiogenic molecules,” such as bFGF (basicfibroblast growth factor); (2) neutralizers of angiogenic molecules,such as an anti-βbFGF antibodies; and (3) inhibitors of endothelial cellresponse to angiogenic stimuli, including collagenase inhibitor,basement membrane turnover inhibitors, angiostatic steroids,fungal-derived angiogenesis inhibitors, platelet factor 4,thrombospondin, arthritis drugs such as D-penicillamine and goldthiomalate, vitamin D3 analogs, alpha-interferon, and the like. Foradditional proposed inhibitors of angiogenesis, see Blood et al., Bioch.Biophys. Acta., 1032:89-118 (1990), Moses et al., Science, 248:1408-1410(1990), Ingber et al., Lab. Invest., 59:44-51 (1988), and U.S. Pat. Nos.5,092,885, 5,112,946, 5,192,744, 5,202,352, and 6,573,256. In addition,there are a wide variety of compounds that can be used to inhibitangiogenesis, for example, peptides or agents that block theVEGF-mediated angiogenesis pathway, endostatin protein or derivatives,lysine binding fragments of angiostatin, melanin or melanin-promotingcompounds, plasminogen fragments (e.g., Kringles 1-3 of plasminogen),tropoin subunits, antagonists of vitronectin αvβ3, peptides derived fromSaposin B, antibiotics or analogs (e.g., tetracycline, or neomycin),dienogest-containing compositions, compounds comprising a MetAP-2inhibitory core coupled to a peptide, the compound EM-138, chalcone andits analogs, and naaladase inhibitors. See, for example, U.S. Pat. Nos.6,395,718, 6,462,075, 6,465,431, 6,475,784, 6,482,802, 6,482,810,6,500,431, 6,500,924, 6,518,298, 6,521,439, 6,525,019, 6,538,103,6,544,758, 6,544,947, 6,548,477, 6,559,126, and 6,569,845.

Depending on the nature of the combinatory therapy, administration ofthe therapeutic antagonists of the invention may be continued while theother therapy is being administered and/or thereafter. Administration ofthe antagonists described herein may be made in a single dose, or inmultiple doses. In some instances, administration of the antagonists iscommenced at least several days prior to the conventional therapy, whilein other instances, administration is begun either immediately before orat the time of the administration of the conventional therapy.

One aspect of the application provides for methods and compositionsuseful in fertility. Decreasing or inhibiting FSH secretion through theadministration of an activin-ActRIIa antagonist is a useful method toinhibit sperm maturation. In females, a decrease of FSH acts to limitproliferation of follicular granulosa cells in the ovary. Decreasing orinhibiting FSH secretion through the administration of anactivin-ActRIIa antagonist is a useful method of contraception. ReducedFSH may also delay the maturation of follicles within the ovary, therebypostponing the maturation of a limited number of follicles in women.Such treatments have the potential for increasing the possibility ofnatural fertilization and pregnancy later in life. Delaying maturationof follicles within the ovary by decreasing FSH secretion is also usefulin preventing the depletion of oocytes, a common side effect ofchemotherapy or similar treatments designed to treat rapidly dividingcells.

The present application also provides for novel compositions comprisingone or more activin-ActRIIa antagonists in combination with one or morecontraceptive agents. Exemplary contraceptive agents include estrogen,progestogen, progestin (e.g., norethynodrel, norethindrone,norgestimate, norgestrel, levonorgestrel, medroxyprogesteroneanddesogestrel), Ormeloxifene (Centchroman)

In certain embodiments, the present invention provides methods oftreating or preventing estrogen related disorders in an individual inneed thereof by administering to the individual a therapeuticallyeffective amount of an activin-ActRIIa antagonist, such as, for example,an ActRIIa polypeptide in order to decrease or inhibit FSH secretion.Because of the controlling function of FSH on estrogen synthesis, thereduction of FSH secretion may also be effective in the treatment ofestrogen related disorders such as uterine fibroids, endometriosis,polycystic ovarian disease, dysfunctional uterine bleeding, and ovariancancer.

7 Pharmaceutical Compositions

In certain embodiments, activin-ActRIIa antagonists (e.g., ActRIIapolypeptides) of the present invention are formulated with apharmaceutically acceptable carrier. For example, an ActRIIa polypeptidecan be administered alone or as a component of a pharmaceuticalformulation (therapeutic composition). The subject compounds may beformulated for administration in any convenient way for use in human orveterinary medicine.

In certain embodiments, the therapeutic method of the invention includesadministering the composition systemically, or locally as an implant ordevice. When administered, the therapeutic composition for use in thisinvention is in a pyrogen-free, physiologically acceptable form.Therapeutically useful agents other than the ActRIIa antagonists whichmay also optionally be included in the composition as described above,may be administered simultaneously or sequentially with the subjectcompounds (e.g., ActRIIa polypeptides) in the methods of the invention.

Typically, ActRIIa antagonists will be administered parentally, andparticularly intravenously or subcutaneously. Pharmaceuticalcompositions suitable for parenteral administration may comprise one ormore ActRIIa polypeptides in combination with one or morepharmaceutically acceptable sterile isotonic aqueous or nonaqueoussolutions, dispersions, suspensions or emulsions, or sterile powderswhich may be reconstituted into sterile injectable solutions ordispersions just prior to use, which may contain antioxidants, buffers,bacteriostats, solutes which render the formulation isotonic with theblood of the intended recipient or suspending or thickening agents.Examples of suitable aqueous and nonaqueous carriers which may beemployed in the pharmaceutical compositions of the invention includewater, ethanol, polyols (such as glycerol, propylene glycol,polyethylene glycol, and the like), and suitable mixtures thereof,vegetable oils, such as olive oil, and injectable organic esters, suchas ethyl oleate. Proper fluidity can be maintained, for example, by theuse of coating materials, such as lecithin, by the maintenance of therequired particle size in the case of dispersions, and by the use ofsurfactants.

In certain embodiments, methods of the invention can be administered fororally, e.g., in the form of capsules, cachets, pills, tablets, lozenges(using a flavored basis, usually sucrose and acacia or tragacanth),powders, granules, or as a solution or a suspension in an aqueous ornon-aqueous liquid, or as an oil-in-water or water-in-oil liquidemulsion, or as an elixir or syrup, or as pastilles (using an inertbase, such as gelatin and glycerin, or sucrose and acacia) and/or asmouth washes and the like, each containing a predetermined amount of anagent as an active ingredient. An agent may also be administered as abolus, electuary or paste.

In solid dosage forms for oral administration (capsules, tablets, pills,dragees, powders, granules, and the like), one or more therapeuticcompounds of the present invention may be mixed with one or morepharmaceutically acceptable carriers, such as sodium citrate ordicalcium phosphate, and/or any of the following: (1) fillers orextenders, such as starches, lactose, sucrose, glucose, mannitol, and/orsilicic acid; (2) binders, such as, for example, carboxymethylcellulose,alginates, gelatin, polyvinyl pyrrolidone, sucrose, and/or acacia; (3)humectants, such as glycerol; (4) disintegrating agents, such asagar-agar, calcium carbonate, potato or tapioca starch, alginic acid,certain silicates, and sodium carbonate; (5) solution retarding agents,such as paraffin; (6) absorption accelerators, such as quaternaryammonium compounds; (7) wetting agents, such as, for example, cetylalcohol and glycerol monostearate; (8) absorbents, such as kaolin andbentonite clay; (9) lubricants, such a talc, calcium stearate, magnesiumstearate, solid polyethylene glycols, sodium lauryl sulfate, andmixtures thereof; and (10) coloring agents. In the case of capsules,tablets and pills, the pharmaceutical compositions may also comprisebuffering agents. Solid compositions of a similar type may also beemployed as fillers in soft and hard-filled gelatin capsules using suchexcipients as lactose or milk sugars, as well as high molecular weightpolyethylene glycols and the like.

Liquid dosage forms for oral administration include pharmaceuticallyacceptable emulsions, microemulsions, solutions, suspensions, syrups,and elixirs. In addition to the active ingredient, the liquid dosageforms may contain inert diluents commonly used in the art, such as wateror other solvents, solubilizing agents and emulsifiers, such as ethylalcohol, isopropyl alcohol, ethyl carbonate, ethyl acetate, benzylalcohol, benzyl benzoate, propylene glycol, 1,3-butylene glycol, oils(in particular, cottonseed, groundnut, corn, germ, olive, castor, andsesame oils), glycerol, tetrahydrofuryl alcohol, polyethylene glycolsand fatty acid esters of sorbitan, and mixtures thereof. Besides inertdiluents, the oral compositions can also include adjuvants such aswetting agents, emulsifying and suspending agents, sweetening,flavoring, coloring, perfuming, and preservative agents.

Suspensions, in addition to the active compounds, may contain suspendingagents such as ethoxylated isostearyl alcohols, polyoxyethylenesorbitol, and sorbitan esters, microcrystalline cellulose, aluminummetahydroxide, bentonite, agar-agar and tragacanth, and mixturesthereof.

The compositions of the invention may also contain adjuvants, such aspreservatives, wetting agents, emulsifying agents and dispersing agents.Prevention of the action of microorganisms may be ensured by theinclusion of various antibacterial and antifungal agents, for example,paraben, chlorobutanol, phenol sorbic acid, and the like. It may also bedesirable to include isotonic agents, such as sugars, sodium chloride,and the like into the compositions. In addition, prolonged absorption ofthe injectable pharmaceutical form may be brought about by the inclusionof agents which delay absorption, such as aluminum monostearate andgelatin.

It is understood that the dosage regimen will be determined by theattending physician considering various factors which modify the actionof the subject compounds of the invention (e.g., ActRIIa polypeptides).The various factors include, but are not limited to, degree of reductionin FSH levels desired, the severity of disease, the patient's age, sex,and diet, the severity of any disease that may be contributing to boneloss, time of administration, and other clinical factors. The additionof other known growth factors to the final composition, may also affectthe dosage. Progress can be monitored by periodic assessment of FSHlevels or other symptoms associated with the FSH-related disorder to betreated.

Experiments with primates and humans have demonstrated that effects ofActRIIa-Fc on FSH are detectable when the compound is dosed at intervalsand amounts sufficient to achieve serum concentrations of about 1000ng/ml, with significant effects on FSH occurring at a dosage of 0.3mg/kg or the equivalent in terms of area-under-curve. In humans, serumlevels of 1000 ng/ml may be achieved with a single dose of 0.3 mg/kg orgreater. The observed serum half-life of the molecule is between about25 and 35 days, substantially longer than most Fc fusion proteins, andthus a sustained effective serum level may be achieved, for example, bydosing with about 0.05 to 0.5 mg/kg on a weekly or biweekly basis, orhigher doses may be used with longer intervals between dosings. Forexample, doses of 0.1, 0.3, 0.5, 0.7, 1, 2 or 3 mg/kg, or values inbetween, might be used on a monthly or bimonthly basis, and the effecton bone may be sufficiently durable that dosing is necessary only onceevery 3, 4, 5, 6, 9, 12 or more months. Longer intervals between dosesare further supported by the duration of the pharmacodynamic effect,which is longer than the duration of drug in the serum. PD effects areobserved for at least 120 days in human patients.

In certain embodiments, the present invention also provides gene therapyfor the in vivo production of ActRIIa polypeptides. Such therapy wouldachieve its therapeutic effect by introduction of the ActRIIapolynucleotide sequences into cells or tissues having the disorders aslisted above. Delivery of ActRIIa polynucleotide sequences can beachieved using a recombinant expression vector such as a chimeric virusor a colloidal dispersion system. Preferred for therapeutic delivery ofActRIIa polynucleotide sequences is the use of targeted liposomes.

Various viral vectors which can be utilized for gene therapy as taughtherein include adenovirus, herpes virus, vaccinia, or, preferably, anRNA virus such as a retrovirus. Preferably, the retroviral vector is aderivative of a murine or avian retrovirus. Examples of retroviralvectors in which a single foreign gene can be inserted include, but arenot limited to: Moloney murine leukemia virus (MoMuLV), Harvey murinesarcoma virus (HaMuSV), murine mammary tumor virus (MuMTV), and RousSarcoma Virus (RSV). A number of additional retroviral vectors canincorporate multiple genes. All of these vectors can transfer orincorporate a gene for a selectable marker so that transduced cells canbe identified and generated. Retroviral vectors can be madetarget-specific by attaching, for example, a sugar, a glycolipid, or aprotein. Preferred targeting is accomplished by using an antibody. Thoseof skill in the art will recognize that specific polynucleotidesequences can be inserted into the retroviral genome or attached to aviral envelope to allow target specific delivery of the retroviralvector containing the ActRIIa polynucleotide. In a preferred embodiment,the vector is targeted to bone or cartilage.

Alternatively, tissue culture cells can be directly transfected withplasmids encoding the retroviral structural genes gag, pol and env, byconventional calcium phosphate transfection. These cells are thentransfected with the vector plasmid containing the genes of interest.The resulting cells release the retroviral vector into the culturemedium.

Another targeted delivery system for ActRIIa polynucleotides is acolloidal dispersion system. Colloidal dispersion systems includemacromolecule complexes, nanocapsules, microspheres, beads, andlipid-based systems including oil-in-water emulsions, micelles, mixedmicelles, and liposomes. The preferred colloidal system of thisinvention is a liposome. Liposomes are artificial membrane vesicleswhich are useful as delivery vehicles in vitro and in vivo. RNA, DNA andintact virions can be encapsulated within the aqueous interior and bedelivered to cells in a biologically active form (see e.g., Fraley, etal., Trends Biochem. Sci., 6:77, 1981). Methods for efficient genetransfer using a liposome vehicle, are known in the art, see e.g.,Mannino, et al., Biotechniques, 6:682, 1988. The composition of theliposome is usually a combination of phospholipids, usually incombination with steroids, especially cholesterol. Other phospholipidsor other lipids may also be used. The physical characteristics ofliposomes depend on pH, ionic strength, and the presence of divalentcations.

Examples of lipids useful in liposome production include phosphatidylcompounds, such as phosphatidylglycerol, phosphatidylcholine,phosphatidylserine, phosphatidylethanolamine, sphingolipids,cerebrosides, and gangliosides. Illustrative phospholipids include eggphosphatidylcholine, dipalmitoylphosphatidylcholine, and distearoylphosphatidylcholine. The targeting of liposomes is also possiblebased on, for example, organ-specificity, cell-specificity, andorganelle-specificity and is known in the art.

EXEMPLIFICATION

The invention now being generally described, it will be more readilyunderstood by reference to the following examples, which are includedmerely for purposes of illustration of certain embodiments andembodiments of the present invention, and are not intended to limit theinvention.

Example 1 ActRIIa-Fc Fusion Proteins

Applicants constructed a soluble ActRIIa fusion protein that has theextracellular domain of human ActRIIa fused to a human or mouse Fcdomain with a minimal linker in between. The constructs are referred toas ActRIIa-hFc and ActRIIa-mFc, respectively.

ActRIIa-hFc is shown below as purified from CHO cell lines (SEQ ID NO:7):

ILGRSETQECLFFNANWEKDRTNQTGVEPCYGDKDKRRHCFATWKNISGSIEIVKQGCWLDDINCYDRTDCVEKKDSPEVYFCCCEGNMCNEKFSYFPEMEVTQPTSNPVTPKPPTGGGTHTCPPCPAPELLGGPSVFLEPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPVPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

The ActRIIa-hFc and ActRIIa-mFc proteins were expressed in CHO celllines. Three different leader sequences were considered:

(i) Honey bee mellitin (HBML): (SEQ ID NO: 8) MKFLVNVALVFMVVYISYIYA (ii) Tissue Plasminogen Activator (TPA): (SEQ ID NO: 9)MDAMKRGLCCVLLLCGAVFVSP  (iii) Native:  (SEQ ID NO: 10)MGAAAKLAFAVFLISCSSGA.

The selected form employs the TPA leader and has the followingunprocessed amino acid sequence:

(SEQ ID NO: 13) MDAMKRGLCCVLLLCGAVFVSPGAAILGRSETQECLFFNANWEKDRTNQTGVEPCYGDKDKRRHCFATWKNISGSIEIVKQGCWLDDINCYDRTDCVEKKDSPEVYFCCCEGNMCNEKFSYFPEMEVTQPTSNPVTPKPPTGGGTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPVPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVM HEALHNHYTQKSLSLSPGK 

This polypeptide is encoded by the following nucleic acid sequence:

(SEQ ID NO: 14) ATGGATGCAATGAAGAGAGGGCTCTGCTGTGTGCTGCTGCTGTGTGGAGCAGTCTTCGTTTCGCCCGGCGCCGCTATACTTGGTAGATCAGAAACTCAGGAGTGTCTTTTTTTAATGCTAATTGGGAAAAAGACAGAACCAATCAAACTGGTGTTGAACCGTGTTATGGTGACAAAGATAAACGGCGGCATTGTTTTGCTACCTGGAAGAATATTTCTGGTTCCATTGAATAGTGAAACAAGGTTGTTGGCTGGATGATATCAACTGCTATGACAGGACTGATTGTGTAGAAAAAAAAGACAGCCCTGAAGTATATTTCTGTTGCTGTGAGGGCAATATGTGTAATGAAAAGTTTTCTTATTTTCCGGAGATGGAAGTCACACAGCCCACTTCAAATCCAGTTACACCTAAGCCACCCACCGGTGGTGGAACTCACACATGCCCACCGTGCCCAGCACCTGAACTCCTGGGGGGACCGTCAGTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGACCCCTGAGGTCACATGCGTGGTGGTGGACGTGAGCCACGAAGACCCTGAGGTCAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTACAACAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAATGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGCCCTCCCAGTCCCCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGTACACCCTGCCCCCATCCCGGGAGGAGATGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTATAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACACGCAGAAGAGCCTCTCCCTGTCTCCGG GTAAATGAGAATTC 

Both ActRIIa-hFc and ActRIIa-mFc were remarkably amenable to recombinantexpression. As shown in FIG. 1, the protein was purified as a single,well-defined peak of protein. N-terminal sequencing revealed a singlesequence of -ILGRSETQE (SEQ ID NO: 11). Purification could be achievedby a series of column chromatography steps, including, for example,three or more of the following, in any order: protein A chromatography,Q sepharose chromatography, phenylsepharose chromatography, sizeexclusion chromatography, and cation exchange chromatography. Thepurification could be completed with viral filtration and bufferexchange. The ActRIIa-hFc protein was purified to a purity of >98% asdetermined by size exclusion chromatography and >95% as determined bySDS PAGE.

ActRIIa-hFc and ActRIIa-mFc showed a high affinity for ligands,particularly activin A. GDF-11 or Activin A (“ActA”) were immobilized ona Biacore CM5 chip using standard amine coupling procedure. ActRIIa-hFcand ActRIIa-mFc proteins were loaded onto the system, and binding wasmeasured. ActRIIa-hFc bound to activin with a dissociation constant(K_(D)) of 5×10⁻¹², and the protein bound to GDF11 with a K_(D) of9.96×10⁻⁹. See FIG. 2. ActRIIa-mFc behaved similarly.

An A-204 Reporter Gene Assay was used to evaluate the effects ofActRIIa-hFc proteins on signaling by GDF-11 and Activin A. Cell line:Human Rhabdomyosarcoma (derived from muscle). Reporter vector:pGL3(CAGA)12 (Described in Dennler et al, 1998, EMBO 17: 3091-3100.) SeeFIG. 3. The CAGAl2 motif is present in TGF-Beta responsive genes (PAI-1gene), so this vector is of general use for factors signaling throughSmad2 and 3.

Day 1: Split A-204 cells into 48-well plate.

Day 2: A-204 cells transfected with 10 μg pGL3(CAGA)12 or pGL3(CAGA)12(10 μg)+pRLCMV (1 μg) and Fugene.

Day 3: Add factors (diluted into medium+0.1% BSA). Inhibitors need to bepreincubated with Factors for 1 hr before adding to cells. 6 hrs later,cells rinsed with PBS, and lyse cells.

This is followed by a Luciferase assay. Typically in this assay, in theabsence of any inhibitors, Activin A shows roughly 10 fold stimulationof reporter gene expression and an ED50˜2 ng/ml. GDF-11: 16 foldstimulation, ED50: ˜1.5 ng/ml. GDF-8 shows an effect similar to GDF-11.

As shown in FIG. 4, ActRIIa-hFc and ActRIIa-mFc inhibit GDF-8 mediatedsignaling at picomolar concentrations. As shown in FIG. 5, threedifferent preparations of ActRIIa-hFc inhibited GDF-11 signaling with anIC50 of approximately 200 pM.

The ActRIIa-hFc was very stable in pharmacokinetic studies. Rats weredosed with 1 mg/kg, 3 mg/kg or 10 mg/kg of ActRIIa-hFc protein andplasma levels of the protein were measured at 24, 48, 72, 144 and 168hours. In a separate study, rats were dosed at 1 mg/kg, 10 mg/kg or 30mg/kg. In rats, ActRIIa-hFc had an 11-14 day serum half life andcirculating levels of the drug were quite high after two weeks (11μg/ml, 110 μg/ml or 304 μg/ml for initial administrations of 1 mg/kg, 10mg/kg or 30 mg/kg, respectively.) In cynomolgus monkeys, the plasma halflife was substantially greater than 14 days and circulating levels ofthe drug were 25 μg/ml, 304 μg/ml or 1440 μg/ml for initialadministrations of 1 mg/kg, 10 mg/kg or 30 mg/kg, respectively.Preliminary results in humans suggests that the serum half life isbetween about 20 and 30 days.

Example 2 ActRIIa-mFc Promotes Bone Growth In Vivo

Normal female mice (BALB/c) were dosed with ActRIIa-mFc at a level of 1mg/kg/dose, 3 mg/kg/dose or 10 mg/kg/dose, with doses given twiceweekly. Bone mineral density and bone mineral content were determined byDEXA, see FIG. 6.

In BALB/c female mice, DEXA scans showed a significant increase (>20%)in bone mineral density and content as a result of ActRIIa-mFctreatment. See FIGS. 7 and 8.

Thus, antagonism of ActRIIa caused increased bone density and content innormal female mice. As a next step, the effect of ActRIIa-mFc on bone ina mouse model for osteoporosis was tested.

Andersson et al. (2001), established that ovariectomized mice sufferedsubstantial bone loss (roughly 50% loss of trabecular bone six weekspost-operation), and that bone loss in these mice could be correctedwith candidate therapeutic agents, such as parathyroid hormone.

Applicants used C57BL6 female mice that were ovariectomized (OVX) orsham operated at 4-5 weeks of age. Eight weeks after surgery, treatmentwith ActRIIa-mFc (10 mg/kg, twice weekly) or control (PBS) wasinitiated. Bone density was measured by CT scanner.

As shown in FIG. 9, untreated, ovariectomized mice showed substantialloss of trabecular bone density relative to the sham controls after sixweeks. ActRIIa-mFc treatment restored bone density to the level of thesham operated mice. At 6 and 12 weeks of the treatment, ActRIIa-mFccaused substantial increase in trabecular bone of OVX mice. See FIG. 10.After 6 weeks of treatment, bone density increased by 24% relative toPBS controls. After 12 weeks, the increase was 27%.

In the sham operated mice, ActRIIa-mFc also caused a substantialincrease in trabecular bone. See FIG. 11. After 6 and 12 weeks, thetreatment produced a 35% increase relative to controls.

In an additional set of experiments, ovariectomized (OVX) or shamoperated mice as described above were treated with ActRIIa-mFc (10mg/kg, twice weekly) or control (PBS) over twelve weeks. Similar to theresults described above for ActRIIa-mFc, OVX mice receiving ActRIIa-mFcexhibited an increase in trabecular bone density of 15% by as early asfour weeks and 25% after 12 weeks of treatment (FIG. 12). Sham operatedmice receiving ActRIIa-mFc similarly showed an increase in trabecularbone density of 22% by as early as four weeks and of 32% after 12 weeksof treatment (FIG. 13).

After twelve weeks of treatment with ActRIIa-mFc, whole body and ex vivofemur DEXA analysis showed that treatment induces an increase in bonedensity in both ovariectomized and sham operated mice (FIGS. 14A and14B, respectively). These results are also supported by ex vivo pQCTanalysis of the femoral midshaft which demonstrated a significantincrease in both total and cortical bone density after twelve weeks oftreatment with ActRIIa-mFc. Vehicle-treated control ovariectomized miceexhibited bone densities that were comparable to vehicle-treated controlsham operated mice (FIG. 15). In addition to bone density, bone contentincreased following ActRIIa-mFC treatment. Ex vivo pQCT analysis of thefemoral midshaft demonstrated a significant increase in both total andcortical bone content after twelve weeks of treatment with ActRIIa-mFcwhile both ovariectomized and sham operated vehicle control-treated miceexhibited comparable bone content (FIG. 16). Ex vivo pQCT analysis ofthe femoral midshaft also showed that ActRIIa-mFc treated mice did notshow a change in periosteal circumference; however ActRIIa-mFc treatmentresulted in a decrease in endosteal circumference indicating an increasein cortical thickness due to growth on the inner surface of the femur(FIG. 17).

Mechanical testing of femurs determined that ActRIIa-mFc was able toincrease the extrinsic characteristics of the bone (maximal load,stiffness and energy to break) which contributed to a significantincrease in the intrinsic properties (ultimate strength) of the bones.Ovariectomized mice treated with ActRIIa-mFc exhibited increased bonestrength to levels beyond sham operated, vehicle treated controls,indicating a complete reversal of the osteoporotic phenotype (FIG. 18).

These data demonstrate that an activin-ActRIIa antagonist can increasebone density in normal female mice and, furthermore, correct defects inbone density, bone content, and ultimately bone strength, in a mousemodel of osteoporosis.

In a further set of experiments, mice were ovariectomized or shamoperated at 4 weeks, and beginning at 12 weeks received either placeboor ActRIIa-mFc (2 times/week, 10 mg/kg) (also referred to as RAP-11 inFIGS. 19-24), for a further period of 12 weeks. A variety of boneparameters were evaluated. As shown in FIG. 19, ActRIIa-mFc increasedvertebral trabecular bone volume to total volume ratios (BV/TV) in boththe OVX and SHAM operated mice. ActRIIa-mFc also improved the trabeculararchitecture (FIG. 20), increased cortical thickness (FIG. 21) andimproved bone strength (FIG. 22). As shown in FIG. 23, ActRIIa-mFcproduced desirable effects at a range of doses from 1 mg/kg to 10 mg/kg.

Bone histomorphometry was conducted at a 2 week time point in shamoperated mice. These data, presented in FIG. 24, demonstrate thatActRIIa-mFc has a dual effect, both inhibiting bone resorption andpromoting bone growth. Thus ActRIIa-mFc stimulates bone growth (anaboliceffect) and inhibits bone resorption (anti-catabolic effect). BV=Bonevolume; TV=total tissue volume. BV/TV is a measure of the percentage ofbone volume that is mineralized. ES=Eroded surface; BS=Bone surface.ES/BS is a measure of bone erosion, and the decrease caused by RAP-011demonstrates an anti-resorptive or anti-catabolic effect. Ms/Bs is themineralizing surface/bone surface ratio, which is an indicator of bonegrowth, or anabolic effect. Similarly, mineral apposition rate (MAR) andbone formation rate per bone surface per day (BFR/BSd) indicate bonegrowth. Measures of osteoblasts (Nob/BPm) and osteoclasts (Noc/BPm) aretaken in order to probe the mechanism of action.

A second bone histomorphometry experiment was conducted in femaleC57BL/6 mice, beginning at an age of twelve weeks. Mice were dosedintraperitoneally twice per week with 10 mg/kg ActRIIa-mFc for twoweeks, four weeks, eight weeks or twelve weeks. Each group wassacrificed five days after the last dose and bones taken for analysis.Mice were calcein labeled nine days and two days prior to euthanasia. Asshown in FIG. 25, the metrics show that ActRIIa-mFc promotes bone growthand mineralization and has both anabolic and anti-catabolic effects. Seefor example the BV/TV ratio, the ES/BS ratio and the MS/BS ratio. Theanabolic effects appear to persist throughout the dosing regimen, whilethe anti-resorptive effects appear to be shorter lived in the mice.

Example 3 ActRIIa-mFc Ameliorates or Prevents Bone Damage in a MurineModel of Multiple Myeloma

Multiple myeloma patients exhibit a bone loss disorder characterized byincreased osteoclast activity and decreased bone formation byosteoblasts. The 5T2MM model of myeloma in mice is based on the use oftumor cells (5T2MM cells) from a type of spontaneous tumor that developsin aged mice and causes effects in mice that are similar to those seenin human multiple myeloma patients. See, e.g., Vanderkerken et al.,Methods Mol Med. 2005; 113:191-205. ActRIIa-mFc was tested for effectsin this model.

5T2MM cells injected into C57B1/KaLwRij mice promoted an increase inosteoclast surface, the formation of osteolytic lesions and caused adecrease in bone area. Bone disease was associated with a decrease inosteoblast number, osteoblast surface and a reduction in mineralization.

Mice bearing 5T2MM cells were treated with ActRIIa-mFc (RAP-011) (10mg/kg, i.p. twice weekly), or a vehicle, from the time of 5T2MMinjection, for a total of 12 weeks. MicroCT analysis of the proximaltibia and lumbar vertebrae demonstrated a 39% and 21% reduction incancellous bone volume (p<0.001 and p<0.01) and a 37% and 15% reductionin trabecular number (p<0.01 and p<0.05) in 5T2MM-bearing mice comparedto naïve mice. RAP-011 completely prevented 5T2MM-induced decreases intrabecular volume and number in both tibia (p<0.001 and p<0.05) andvertebrae (p<0.01 and p<0.05) when compared to vehicle treated mice.Bone volume was 19% higher in the tibia (p=168) and 12% higher invertebrae (p<0.05) of RAP-011 treated mice when compared to naïve mice.RAP-011 prevented the development of osteolytic bone lesions (p<0.05).This effect is illustrated in FIG. 26. While a preliminary assessment ofthe data failed to identify significant effects on serum paraprotein (abiomarker of multiple myeloma tumor cells) or myeloma burden in thisstudy, a further analysis indicated that serum paraprotein wassubstantially decreased in all but one of the treated animals, andfurther that the volume of healthy bone marrow was substantiallyincreased, indicating a decrease in the myeloma tumor cell burden.

Therefore, ActRIIa-mFc may be used to decrease the effects of bonedisease resulting from multiple myeloma and to treat the tumor cellsthemselves.

Example 4 Characterization of an ActRIIa-hFc Protein

ActRIIa-hFc fusion protein was expressed in stably transfected CHO-DUKXB11 cells from a pAID4 vector (SV40 ori/enhancer, CMV promoter), using atissue plasminogen leader sequence of SEQ ID NO:9. The protein, purifiedas described above in Example 1, had a sequence of SEQ ID NO:7. The Fcportion is a human IgG1 Fc sequence, as shown in SEQ ID NO:7. Sialicacid analysis showed that the protein contained, on average, betweenabout 1.5 and 2.5 moles of sialic acid per molecule of ActRIIa-hFcfusion protein.

This purified protein showed a remarkably long serum half-life in allanimals tested, including a half-life of 25-32 days in human patients(see Example 6, below). Additionally, the CHO cell expressed materialhas a higher affinity for activin B ligand than that reported for anActRIIa-hFc fusion protein expressed in human 293 cells (del Re et al.,J Biol Chem. 2004 Dec. 17; 279(51):53126-35.) Additionally, the use ofthe tPa leader sequence provided greater production than other leadersequences and, unlike ActRIIa-Fc expressed with a native leader,provided a highly pure N-terminal sequence. Use of the native leadersequence resulted in two major species of ActRIIa-Fc, each having adifferent N-terminal sequence.

Example 5 Human Clinical Trial

The protein described in Example 5 was administered to human patients ina randomized, double-blind, placebo-controlled study that was conductedto evaluate, primarily, the safety of the protein in healthy,postmenopausal women. Forty-eight subjects were randomized in cohorts of6 to receive either a single dose of ActRIIa-hFc or placebo (5 active:1placebo). Dose levels ranged from 0.01 to 3.0 mg/kg intravenously (IV)and 0.03 to 0.1 mg/kg subcutaneously (SC). All subjects were followedfor 120 days. Subjects were excluded from study participation if theytook medications affecting bone metabolism within 6 months of studyentry. Safety evaluations were conducted following each cohort todetermine dose escalation. In addition to pharmacokinetic (PK) analyses,the biologic activity of ActRIIa-hFc was also assessed by measurement ofbiochemical markers of bone formation and resorption, and FSH levels.

No serious adverse events were reported in this study. Adverse events(AEs) were generally mild and transient. Preliminary analysis of AEsincluded headache, elevated laboratory values, cold symptoms, emesis orvomiting, intravenous infiltration, and hematoma at injection site.

PK analysis of ActRIIa-hFc displayed a linear profile with dose, and amean half-life of approximately 25-32 days. The area-under-curve (AUC)for ActRIIa-hFc was linearly related to dose, and the absorption afterSC dosing was essentially complete (see FIGS. 27 and 28). These dataindicate that SC is a desirable approach to dosing because it providesequivalent bioavailability and serum-half life for the drug whileavoiding the spike in serum concentrations of drug associated with thefirst few days of IV dosing (see FIG. 28). ActRIIa-hFc caused a rapid,sustained dose-dependent increase in serum levels of bone-specificalkaline phosphatase (BAP), which is a marker for anabolic bone growth,and a dose-dependent decrease in C-terminal type 1 collagen telopeptideand tartrate-resistant acid phosphatase 5b levels, which are markers forbone resorption. Other markers, such as P1NP showed inconclusiveresults. BAP levels showed near saturating effects at the highest dosageof drug, indicating that half-maximal effects on this anabolic bonebiomarker could be achieved at a dosage of 0.3 mg/kg, with increasesranging up to 3 mg/kg. Calculated as a relationship of pharmacodynamiceffect to AUC for drug, the EC50 is 51,465 (day*ng/ml). See FIG. 29.These bone biomarker changes were sustained for approximately 120 daysat the highest dose levels tested. There was also a dose-dependentdecrease in serum FSH levels consistent with inhibition of activin.Substantial decreases in FSH levels were observed with doses ofActRIIa-hFc ranging from 0.10 mg/kg up to 3 mg/kg. Decreases in mean FSHlevels of 30-40% were observed with 1 and 3 mg/kg dosing, and inindividual patients at the 3 mg/kg dose, decreases of up to 50% of FSHrelative to baseline were observed. It should be noted thatpost-menopausal women exhibit a relatively consistent elevated FSHlevel, making it relatively easy to observe the effects of the drug onFSH. In men and reproductively active women, the baseline FSH level mayvary widely making it difficult to assess the specific degree ofinhibition, but nonetheless, the activin-FSH signaling axis is intact inthese individuals and it is expected that ActRIIa-hFc will inhibit FSHproduction to a significant degree even if it is difficult to quantifythe effect on FSH in these populations. Calculated as a relationship ofpharmacodynamic effect to AUC for drug with respect to the effect onFSH, the EC50 is approximately 250,000 (day*ng/ml). See FIG. 32.

A single dose of ActRIIa-hFc given to healthy postmenopausal women wassafe and well-tolerated for the range of dose levels tested. Theprolonged PK and pharmacodynamic effects suggest that intermittentdosing would be appropriate for future studies. For example, dosing onthe basis of serum half-life could be performed on a monthly basis, oron the order of once every two, three, four, five or six weeks.Additionally, because the pharmacodynamic effect extends far beyond theserum residence of the drug, dosing could be performed on the basis ofthe pharmacodynamic effect, meaning that dosing every three months orevery two, three, four, five, six or even twelve months may be effectiveto produce the desired effect in patients. This clinical trialdemonstrates that, in humans, ActRIIa-hFc is an osteoanabolic agent withbiological evidence of both an increase in bone formation and a decreasein bone resorption.

Example 6 Co-Administration of ActRIIa-mFc and a Bisphosphonate

Bisphosphonates are a class of drugs that are widely used to treatdisorders associated with low bone mineral density, includingosteoporosis and cancer-related bone loss. Bisphosphonates have a potentanti-resorptive activity, inhibiting osteoclasts. Perhaps becauseosteoclasts are required both for bone breakdown and bone growth,bisphosphonates appear to diminish the effects of parathyroid hormone(PTH), one of the only known anabolic bone growth agents (Black et al.,N Engl J Med. 2003 Sep. 25; 349(13):1207-15; Samadfam et al.,Endocrinology. 2007 June; 148(6):2778-87.)

To test the utility of ActRIIa-Fc treatment in patients that hadpreviously or were concomitantly receiving bisphosphonate or otheranti-resorptive therapy, mice were tested with combined ActRIIa-mFc andzoledronate, a bisphosphonate compound. 12 week old C57BL/6N mice weretreated as follows:

Group 1 PBS

Group 2 ActRIIa-mFc (RAP-011) (10 mg/kg) twice per week (with Group 3and 4)Group 3 Zoledronic Acid (ZOL) singe dose (20 mg/kg)Group 4 ZOL (1 dose), 3 days later ActRIIa-mFc (RAP-011) (1 mg/kg) twiceper weekGroup 5 ZOL (1 dose), 3 days later ActRIIa-mFc (RAP-011) (10 mg/kg)twice per weekTotal BMD was determined by DEXA scan (PIXI) prior to dosing and at 3and 8 weeks of treatment.

As shown in FIG. 30, total BMD increased markedly in all treatmentgroups, with the combination of ZOL and ActRIIa-mFc producing thegreatest effects. These results indicate that ActRIIa-Fc proteins can beused to increase bone density, even in patients that have receivedbisphosphonate therapy.

Example 7 Alternative ActRIIa-Fc Proteins

A variety of ActRIIa variants that may be used according to the methodsdescribed herein are descried in the International Patent Applicationpublished as WO2006/012627 (see e.g., pp. 55-58), incorporated herein byreference in its entirety. An alternative construct may have a deletionof the C-terminal tail (the final 15 amino acids of the extracellulardomain of ActRIIa. The sequence for such a construct is presented below(Fc portion underlined)(SEQ ID NO: 12):

ILGRSETQECLFFNANWEKDRTNQTGVEPCYGDKDKRRHCFATWKNISGSIEIVKQGCWLDDINCYDRTDCVEKKDSPEVYFCCCEGNMCNEKFSYFPEMTGGGTHTCPPCPAPELLGGPSVFLEPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPVPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

INCORPORATION BY REFERENCE

All publications and patents mentioned herein are hereby incorporated byreference in their entirety as if each individual publication or patentwas specifically and individually indicated to be incorporated byreference.

While specific embodiments of the subject matter have been discussed,the above specification is illustrative and not restrictive. Manyvariations will become apparent to those skilled in the art upon reviewof this specification and the claims below. The full scope of theinvention should be determined by reference to the claims, along withtheir full scope of equivalents, and the specification, along with suchvariations.

We claim:
 1. A method for inhibiting FSH production in a patient desiring to delay or inhibit his or her germ cell maturation, the method comprising administering an amount of ActRIIa-Fc fusion protein effective to reduce FSH activity in the subject, wherein the ActRIIa-Fc fusion protein comprises an amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO:3.
 2. The method of claim 1, wherein the ActRIIa-Fc fusion protein comprises an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO:3.
 3. The method of claim 1, wherein the ActRIIa-Fc fusion protein comprises the amino acid sequence of SEQ ID NO:3.
 4. The method of claim 1, wherein the ActRIIa-Fc fusion protein comprises the amino acid sequence of SEQ ID NO:2.
 5. The method of claim 1, wherein the ActRIIa-Fc fusion protein is a dimer formed of two polypeptides that each comprise an amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO:2.
 6. The method of claim 5, wherein each polypeptide of the dimer comprises the amino acid sequence of SEQ ID NO:2.
 7. The method of claim 6, wherein the ActRIIa-Fc fusion protein comprises three or more sialic acid moieties.
 8. The method of claim 7, wherein the ActRIIa-Fc fusion protein is produced by expression in CHO cells.
 9. The method of claim 7, wherein the ActRIIa-Fc fusion protein comprises between three and five sialic acid moieties.
 10. The method of claim 1, wherein the ActRIIa-Fc fusion protein has an amino acid sequence of SEQ ID NO:7.
 11. The method of claim 6, wherein the ActRIIa-Fc fusion protein has a serum half-life of 25 to 32 days on average in normal, healthy humans and equivalent bioavailability when administered intravenously or subcutaneously.
 12. The method of claim 11, wherein the ActRIIa-Fc fusion protein has four sialic acid moieties per dimer. 